Vascular cognitive impairment and dementia (VCID) include a wide spectrum of chronic manifestations of vascular disease related to large vessel strokes and small vessel disease (SVD). Lacunar strokes and white matter (WM) injury are consequences of SVD. The main vascular risk factor for SVD is brain hypoperfusion from cerebral blood vessel narrowing due to chronic hypertension. The hypoperfusion leads to activation and degeneration of astrocytes with the resulting fibrosis of the extracellular matrix (ECM). Elasticity is lost in fibrotic cerebral vessels, reducing the response of stiffened blood vessels in times of increased metabolic need. Intermittent hypoxia/ischaemia activates a molecular injury cascade, producing an incomplete infarction that is most damaging to the deep WM, which is a watershed region for cerebral blood flow. Neuroinflammation caused by hypoxia activates microglia/macrophages to release proteases and free radicals that perpetuate the damage over time to molecules in the ECM and the neurovascular unit (NVU). Matrix metalloproteinases (MMPs) secreted in an attempt to remodel the blood vessel wall have the undesired consequences of opening the blood–brain barrier (BBB) and attacking myelinated fibres. This dual effect of the MMPs causes vasogenic oedema in WM and vascular demyelination, which are the hallmarks of the subcortical ischaemic vascular disease (SIVD), which is the SVD form of VCID also called Binswanger's disease (BD). Unravelling the complex pathophysiology of the WM injury-related inflammation in the small vessel form of VCID could lead to novel therapeutic strategies to reduce damage to the ECM, preventing the progressive damage to the WM.
- Binswanger's disease
- extracellular matrix
- matrix metalloproteinases
- vascular cognitive impairment
The prevalence of vascular diseases is projected to continue to increase with people living into the eighth and ninth decades, which will result in an increase in patients with dementia due to old age. The confluence of aging, vascular diseases and neurodegeneration will result in a dramatic increase in the numbers of people with vascular cognitive impairment and dementia (VCID) . Blood vessel disease damages the brain through three mechanisms: (1) restriction of cerebral blood flow, reducing delivery of essential nutrients including, oxygen, glucose and amino acids; (2) ischaemic/hypoxic injury secondary to thromboses and emboli from the carotids and heart; and (3) inflammation in and around blood vessels damaged by hypertension, diabetes, hyperlipidaemia and other vascular risk factors. Recently, much interest has focused on treatment of vascular risk factors because damage to the blood vessels causes neuroinflammation, which leads to progressive damage to the deep white matter (WM). Hypertension alters the structure of blood vessels, particularly the arterioles that deliver blood into the central regions of the brain . Remodelling of the blood vessels as a result of the hypertension alters both the vessel wall and the surrounding extracellular matrix (ECM) . The complex interaction of the ECM with matrix-degrading proteases plays an important role in determining the extent and type of damage that occurs to the blood vessels and surrounding ECM . There is increasing understanding of the complex interaction between the injured vessels and the surrounding ECM both in acute and chronic injury and this knowledge can aid in the planning of interventions to promote recovery. Large vessel strokes are sporadic events with an unpredictable natural history, which complicate clinical trials, and several recent reviews have addressed this form of VCID [5–7]. The focus of this review is to describe the progressive form of VCID due to small vessel disease (SVD) that leads to extensive WM damage, and due to the progressive nature, lends itself to treatment trials. The small vessel form of VCID is referred to as subcortical ischaemic vascular disease (SIVD), or when there is mainly WM involvement, the term Binswanger's disease (BD) is often used. Although BD has been variously defined in the literature, in this review, it will refer to the non-hereditary, non-immunological form of protease-mediated inflammatory demyelination as opposed to other forms of vascular disease leading to WM damage.
Vascular cognitive impairment dementia
Definitions of vascular causes of dementia have evolved over the past several years [6,8,9]. Initially dementia was thought to be related to the volume of multiple strokes, and the condition was called multi-infarct dementia (MID) . With the advent of modern brain imaging, which revealed a high incidence of WM damage not necessarily related to strokes, there was a growing awareness of the importance of slowly progressive changes in the brain related to SVD . It is now thought by many investigators that SVD is the major cause of VCID, and that there are a large number of patients with both SVD and Alzheimer's disease (AD), the so-called mixed dementia, which is commonly found pathologically [12–16].
Both large and small vessel disease affect the brain's ECM, but the major changes are seen with SVD. Recent studies have begun to unravel the role of ECM in normal and pathological conditions. ECM is important in maintaining normal function of blood vessels, remodelling of synapses, and the control of movement of interstitial fluid (ISF) through the brain. The ISF and cerebrospinal fluid (CSF) are continuous and form one fluid that plays a role in delivery of nutrients and removal of metabolic waste . The important role of the CSF/ISF to act as the brain's “lymphatic fluid”, was recognized by early investigators who called this the Third Circulation . ISF is secreted by cerebral blood vessels, and it drains into the CSF through the WM by bulk flow and along perivascular spaces [19–21]. Drainage of CSF/ISF across the cribriform plate into the cervical lymphatics is a route for fragments of myelin and of the ECM molecules to reach the general circulation, where they can cause an immunological reaction [22–24]. Recent studies in mouse brain have demonstrated CSF/ISF drainage along basal lamina and perivascular spaces .
The changes in blood vessels leads to hypoperfusion and hypoxia mainly affect the vulnerable, watershed regions of the deep WM, producing abnormalities that can be visualized on brain MRI using fluid-attenuated inversion recovery (FLAIR) sequences [26–29]. These changes occur over long periods of time and eventually damage sufficient amounts of brain tissue to produce symptoms consistent with WM injury, including small stroke-like episodes, imbalance, and impairment of executive function. In addition, there is growing evidence that deposits of proteins associated with AD, such as amyloid-β1–42 (Aβ) and phosphorylated tau181 (pTau), are increased when there is damage to the blood vessels .
Hypertension increases blood vessel fibrosis, leading to changes in type 4 collagen and other ECM molecules with stiffening of the vessel wall, impairing cerebral blood flow, particularly at times of increased need . These changes in the vessel wall and ECM require 10 or more years, allowing for compensatory mechanisms in the early stages, such as augmentation of collateral flow, that are finally overwhelmed as the process reaches a critical point . The deep WM has a watershed-like blood supply, making it more vulnerable to reduction in cerebral blood flow than other brain regions 
Changes in the blood vessels and brains of hypertensive patients begin early in life and often progress silently, requiring many years to produce clinical symptoms. In a recent study of third generation participants in the long-term Framingham study, patients with hypertension at age 40, as defined by blood pressures of 140/90, were found to have WM changes on diffusion tension imaging (DTI) in normal-appearing WM that were seen in the brain 7 years earlier than those with blood pressures of 120/80 . These longitudinal studies complement the cross-sectional studies showing that white matter hyperintensities (WMHs) on FLAIR MRI are related to cognitive function, which is more evident, using DTI than FLAIR MRI [34,35].
The changes in the WM related to the reduced blood flow without frank infarction have been called “incomplete infarction” and are suggested to be due to the inability of the brain to respond to metabolic needs by increasing the blood flow . Although postulated originally from the results of pathological studies of patients with VCID, the impaired responsivity of blood vessels in SIVD has recently been confirmed experimentally with studies of cerebral blood flow showing that during controlled inhalation of carbon dioxide, a potent vasodilator, the expected dilatation fails to occur; the regions with reduced blood flow during carbon dioxide challenge were shown to correlate with the regions where the WM lesions increased in size .
WMHs on MRI suggest either demyelination or oedema, which appear similar on FLAIR (Figure 1A). However, the presence of WMHs on MRI is non-diagnostic since they occur in a high percentage of normal elderly people . When the WM has undergone pathological changes, WHMs can be differentiated with reduced levels of N-acetylaspartate (NAA) on proton magnetic resonance spectroscopy (1H-MRS) or reduced fractional anisotropy (FA) on DTI [38,39].
Studies in humans show disruption of the blood–brain barrier (BBB) as increased permeability on dynamic contrast-enhanced MRI (DCEMRI) (Figures 1C and 1D) [40,41]. This is a subtle disruption of the neurovascular unit (NVU) that cannot be seen on routine contrast-enhanced MRI, but requires the use of multiple fast T1-weighted images over 20 min with mathematical calculations of blood-to-brain transfer constants obtained with the contrast agent, gadolinium-diethylenetriamine penta-acetic acid (DTPA) [42,43]. Another indicator of a disrupted BBB is an increase in albumin in the CSF, which is often seen in VCID .
Pathophysiology of extracellular matrix changes in VCID
Pathological studies in humans show infiltrating macrophages and activated microglia, which are responding to the changes in the blood vessels and to reduced oxygen, around the fibrotic blood vessels; these inflammatory cells release proteases and free radicals [45–47]. The release of proteases and free radicals by the macrophage/microglia has a dual effect. On the one hand, they directly attack the blood vessel ECM, loosening tight junctions and breaking down the basal lamina. They also attack the myelinated fibres, breaking down the myelin and leading to loss of normal WM function [48–50]. This non-immunological process has been called “by-stander” demyelination, which could explain both the disruption of the BBB by the proteases released by the inflammatory cells and demyelination induced by vascular disease . Matrix metalloproteinases (MMPs) play key roles in both processes when they are released and activated from endogenous and infiltrating macrophages/microglia. Several factors contribute to the release of proteases, including the hypoxic state and the fibrotic vessels, which could trigger the inflammatory response, possibly in an attempt to repair the damaged vessels.
In addition to the reduction in cerebral blood flow over time due to chronically elevated hypertension, occlusion of small blood vessels causes a series of strokes that accumulate over time as lacunar infarcts in WM and basal ganglia. The culmination of the reduction in blood flow along with scattered vessel occlusions is extensive injury to the deep WM, which is exacerbated by the architecture of the cerebral blood vessels that penetrate into the deeper structures from the cortical surface, and by intermittent hypoxia as occurs, for example, during times of increased need for nutrients in sleep apnoea and congestive heart failure.
Pathological studies show the presence of MMPs in brain tissue of patients with VCID [45–47]. MMP-2 is found in astrocytes in the WM, whereas pericytes, surrounding the endothelial cells, immunostain for MMP-3. The presence of MMPs in the inflammatory cells found around blood vessels suggests a role of MMPs in the alterations that occur in the ECM. The actions of MMPs that contribute to the remodelling of the vessel wall, result in changes in the MMPs in the CSF that can be measured in patients with VCID [52–55]
MMPs in the CSF could come from the systemic circulation by crossing the BBB or they could be produced endogenously. It is possible to determine the origin of the MMPs by forming a ratio of the MMPs in each compartment with the albumin in that compartment similar to the IgG index used in the diagnosis of multiple sclerosis. The presence of MMPs in the CSF suggests an active inflammatory process with BBB disruption. In patients with extensive WM injury related to neuroinflammation, the MMP-2 index is reduced and correlated with the albumin index (Qalb), a marker of BBB opening (Figure 2) .
Normally, the constitutive enzyme, MMP-2, is found in the CSF in sufficient quantities to be detectable by gel zymography or ELISA; MMP-2 index is reduced in CSF when inflammation is present. On the other hand, the inducible MMPs, MMP-3 and MMP-9, are barely detectable until there is an injury. In the acute situation, the ischaemic injury leads to the expression of MMP-9 and MMP-3. The situation is more complicated in chronic conditions such as VCID, where the levels of MMP-2 are reduced and MMP-9 is variably expressed; the MMPs are in a latent form, which only provides limited information regarding their action. [52–54,57].
Pathophysiology of VCID explored with studies in animals
Animal models demonstrate the pathophysiology of SIVD . Two animal models have been most studied: bilateral occlusion of the common carotid arteries (BCAO) in the normotensive rat and spontaneously hypertensive stroke prone rats (SHR/SP) with dietary manipulation. A third model was recently reported in rats and mice that involved gradual occlusion of the carotid arteries with a novel ameroid constrictor, but few studies have been reported to date using this method [59,60]. The ECM is altered in these models due to the action of the MMPs. With BCAO there is an increase in MMP-2 that leads to BBB disruption, which can be seen both on MRI and histologically; the BCAO animals show disruption of the BBB 3 days after the occlusion associated with increased expression of MMP-2 . Knockout mice lacking the MMP-2 gene have reduced disruption of the BBB, which is also seen with inhibitors of MMPs .
Animal studies in hypertensive rat models have shown that loss of oxygen initiates a cascade of molecular events, triggering an injury response. Hypoxia inducible factor-1α (HIF-1α) is stabilized by reduction in proteolysis, which frees the dimer to trigger transcription of a large number of genes . The fur gene is induced by HIF-1α, leading to the production of the convertase, furin, which is important because of the large number and variety of bioactive proteins and peptides that can be activated through its activity, including key elements involved in normal and pathophysiological conditions, such as membrane type 1 matrix metalloproteinase (MMP-14) and transforming growth factor-β1 (TGF-β1). Both mediators are well-characterized furin substrates that have been shown to profoundly affect many aspects of injury progression . The slow conversion of normal blood vessels to ones with damage occurs over time as a series of “hits”. The first is the long-standing hypertension, which damages the blood vessels, the second is the hypoxic phase with the initiation of the hypoxia-sensitive genes and finally the third hit is the induction of an inflammatory response with opening of the BBB, disruption of myelin and cell death (Figure 3).
Under normal conditions, the remodelling of the ECM proceeds in an orderly fashion with the activity of MMP-2 constrained to the close vicinity of the blood vessel wall due to the tethering to the cell wall by membrane-type metalloproteinase during activation. Normally, activation of the MMPs is tightly controlled by multiple mechanisms that maintain the latency state (Figure 4). This orderly pattern of activation is lost during a hypoxic episode due to the production of the inflammatory MMPs, MMP-3 and MMP-9, which causes more widespread tissue damage [50,65]. As the molecular injury cascade progresses, cytokines are formed, triggering the transcription of the inducible MMPs that cause the irreversible protein damage. Tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) act at the activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) transcription sites to promote the formation of latent MMP-9 and MMP-3, which are activated by enzymes, free radicals or auto-activated .
Another animal model that is closer to the clinical condition is the SHR/SP . Modifying the diet by reducing the protein and adding salt along with occluding one carotid artery can accelerate the damage to the WM. Studies using the SHR/SP model showed that starting the diet with carotid occlusion after 12 weeks of life results in death by 16 weeks, markedly shortening the normal lifespan of 9 months in these rats . Analysis of the brain tissue shows an increase in HIF-1α, the induction of MMP-9, opening of the BBB and breakdown of myelin . The changes in the WM can be seen with multimodal MR (Figure 5). In the SHR/SP with dietary manipulation, there is hypoxia in the deep WM as shown by EPR, which begins around the 12th week of life when the hypertension reaches a maximum . Treatment with the anti-inflammatory, MMP inhibitor, minocycline, reduces damage to the WM, prolongs life and improves learning in the Morris water maze . Thus, the SHR/SP with dietary manipulation and carotid occlusion is a model that can be used to test drugs for eventual translation into human studies.
Molecular composition of the extracellular matrix
Molecules of the ECM are critical in preserving the structure and function of all central nervous system (CNS) cells. Comprising 10–20% of brain tissue, ECM is organized into three major compartments: basal lamina, perineuronal nets (PNN) and parenchyma (Table 1) . Around the endothelial cells is a basal lamina that separates endothelial cells from brain parenchyma. The basal lamina is part of the NVU, and is made up of type IV collagen, heparan sulfate proteoglycans (HSPG), junctional adhesion complexes, laminin–nidogen (or entactin), fibronectin, dystroglycan and perlecan. Between the cells of the parenchyma is an interstitial matrix that is made up of chondroitin sulfate proteoglycans (CSPG), hyaluronan, tenascin R and link proteins. The third component of the ECM is the PNN, which are a layer of lattice-like matrix that covers the surface of the soma and dendrites. PNN are mainly composed of hyaluronan, CSPG, link proteins and tenascin R, and they play a direct role in the control of CNS plasticity .
Multiple layers make up the NVU . The endothelial cells and the tight junction proteins provide the first layer of protection. Brain endothelial cells use energy from ATP to form ISF, have few pinocytotic vesicles and facilitate transport of glucose and amino acids. Tight junction proteins join the ends of brain endothelial cells forming a barrier to large non-lipophilic molecules . The principle tight junction proteins are occludin, claudin, zonula occudens and junctional adhesion molecules (JAM). Pericytes are embedded in the basal lamina. Finally, astrocytic endfeet form the outer layer [73–76]. The third component of the ECM is the interstitial proteins, which are composed of long proteoglycan chains, which provide structure and retain water.
Matrix metalloproteases and the extracellular matrix
ECM is continuously being produced and broken down. This delicate balance is preserved mainly by the MMPs, which degrade the extracellular proteins under both normal and pathological conditions. The constitutive enzymes, MMP-2 and MMP-14, appear to be more important in the chronic changes induced by hypertension than the inducible enzymes that play a dominant role in acute matrix remodelling, with the exception that MMP-3 which is found in the pericytes. SHR/SP develops WM injury with an increase in MMP-2 in the reactive astrocytes.
The MMPs are normally maintained in a latent state and require activation by free radicals or other proteases in order to act . Once activated, they are quickly neutralized by one of four tissue inhibitors of metalloproteinases (TIMPs) [78,79]. Different cell types form the MMPs. MMP-9 is found in the endothelial cells and in neutrophils. Pericytes produce MMP-3. Microglia cells contain MMP-9 and MMP-3 . Growing blood vessels secrete the MMPs in conjunction with plasminogen activators to breakdown the ECM in their path, permitting them to spread .
The impact of hypertension on blood vessels over extended periods of time alters the vessel wall and the surrounding ECM. Lumens are narrowed; fibrotic outer walls induce an inflammatory response that activates endogenous microglia and recruits circulating macrophages. Macrophages/microglia are activated possibly in an attempt at remodelling the blood vessel wall to strengthen it. However, in the process there is release of destructive proteases that loosen basal lamina and tight junctions in endothelial cells and attack myelinated fibres. As a consequence of the “by-stander” demyelination during this chronic phase, the major features of the pathological changes in the Binswanger form of SIVD are produced (Figure 6).
Recently, MMP-9 was shown to play in a novel role in synaptic remodelling during learning by its action on the ECM PNN around the synaptic structures . ECM is important in homoeostatic processes such as scaling of synaptic responses, synaptic remodelling and stabilization of synaptic connectivity . Rats learning the Morris water maze exhibit hippocampal changes in synaptic morphology and physiology that are required for learning . MMP-3 and -9 increased transiently during water maze acquisition; blocking MMP-3 and -9 with antisense oligonucleotides and an MMP inhibitor altered long-term potentiation and prevented learning in the Morris water maze . Thus, an increase in MMP-9 in the hippocampus is associated with the laying down of memories, presumably by altering long-term potentiation. Although MMP-9 inhibitors reduce injury in acute ischaemia, they are not effective in the chronically hypertensive animals (L. Raz, Y. Yang, and G. Rosenberg, unpublished data). The importance of this finding is that attempts to treat MMP-related brain injury with MMP-9 selective inhibitors in chronic experiments may encounter problems with learning new memories .
Deposition of ECM after an injury causes changes in the interstitial matrix that impedes recovery . Reactive astrocytes are a major source of CSPG that contribute to scar tissue. Deposition of ECM components into the interstitial matrix is influenced by inflammatory cytokines and TGF-β . Activated astrocytes lead to an increase in ECM proteins that inhibits repair and regrowth of axons in WM . An attempt to reduce the scarring of the WM to encourage repair is a major effort in multiple sclerosis research, but very little is known about the repair process in VCID.
Inhibitors of MMPs in treatment of neuroinflammation
A critical factor in planning for the use of MMP inhibitors in treatment of various types of brain injury is to take into account the dual role of MMPs: beneficial during development and recovery for ongoing remodelling of the ECM, but detrimental during the acute stages of an injury. Since both the acute injury phase and the subsequent repair require MMPs, the timing of the administration of the MMP inhibitors is critical. Many MMPs are up-regulated in pathological conditions such as stroke, multiple sclerosis, bacterial meningitis and brain and spinal cord injury. Treatment of the increased levels of MMPs early in the injury cycle has been shown to be beneficial in reducing cytotoxicity, disruption of the BBB and promotion of neuroinflammation . On the other hand, during repair, the MMPs have reparative functions in the post-acute phase of CNS injury, and facilitate angiogenesis and neurogenesis . Blocking MMP-9, which breaks down the matrix to allow for the growth, impairs axonal repair [88,89]. MMPs participate in myelinogenesis as shown by the finding of deficient myelination in the corpus callosum of MMP-9 and/or MMP-12 null mice from postnatal days 7–14 compared with that of wild-type mice; insulin-like growth factor-1 (IGF-1) was involved in the deficient myelination in MMP null mice; the addition of IGF-1 normalized the lack of maturation of oligodendrocytes that occurred in cultures from MMP-12 null mice . In mouse models of spinal cord injury, increased MMP-2 immunoreactivity is linked to greater sparing of WM, reduced glial scarring and improved locomotor recovery .
Although much is known about the role of MMPs in acute injury and repair, relatively little is understood about their role in chronic vascular disease. Minocycline, a tetracycline derivative that has multiple beneficial actions, including reduction in neuroinflammation and inhibition of MMPs, has a beneficial effect in the hypertensive model of VCID where it has been shown to reduce WM damage and improve behaviour during recovery [68,92,93]. A recent report described an alteration in the astrocytes due to degeneration that produced clasmotodendrocytes. Aquaporin-4, the water channel molecule thought to clear oedema fluid and participate in water balance, was shown to be displaced on the astrocyte endfeet. Identifying ways to prevent astroycte changes may offer new therapeutic avenues . Although the use of MMP inhibitors to modify the changes in the ECM has experimental support, much will need to be learned about the multiple roles played by the MMPs before clinical trials are possible.
The studies described in this review signal a new direction for research that involves unravelling the complex molecular chemistry of the ECM, while simultaneously devising new therapeutic approaches based on the new knowledge. Neuroinflammation driven by macrophages/microglia plays a major role in both the breakdown of the ECM and in the repair. It will be critical to determine the time course of both reactions so that therapy can be applied at the optimal time to either block the damaging effects of the proteases and free radicals or promote their beneficial actions. This will require more information on the natural history of the changes occurring in the ECM during injury. Although we have learned a great deal about the role of MMPs, less is known about other proteases and the interaction of the proteases with free radicals. New methods for the analysis of the activity of the proteases show that activity is more important than the presence of the latent forms. Finally, the recently discovered role of the proteases, particularly MMP-9 in the synaptic plasticity, will require more intense study to determine the effect of the anti-inflammatory protease inhibitors on synaptic remodelling during learning.
Abbreviations: Aβ, amyloid-β1–42; AD, Alzheimer's disease; ADC, apparent diffusion coefficient; AP-1, activator protein-1; ASL, arterial spin labelling; BBB, blood–brain barrier; BCAO, bilateral occlusion of the common carotid arteries; BD, Binswanger's disease; CNS, central nervous system; CON, control CSF; COX-2, cyclooxygenase-2; CSF, cerebrospinal fluid; CSPG, chondroitin sulfate proteoglycans; DCEMRI, dynamic contrast-enhanced MRI; DTI, diffusion tension imaging; DTPA, diethylenetriamine penta-acetic acid; ECM, extracellular matrix; EPO, erythropoietin; FA, fractional anisotropy; FLAIR, fluid-attenuated inversion recovery; HIF-1α, hypoxia inducible factor-1α; 1H-MRS, proton magnetic resonance spectroscopy; HSPG, heparan sulfate proteoglycans; IGF-1, insulin-like growth factor-1; IL-1β, interleukin-1β; ISF, interstitial fluid; JAM, junctional adhesion molecules; JDP, Japanese permissive diet; MID, multi-infarct dementia; MMP, matrix metalloproteinase; NAA, N-acetylaspartate; NF-κB, nuclear factor-κB; NVU, neurovascular unit; PHD, pyrrole hydroxylase; PNN, perineuronal nets; pTau, phosphorylated tau181; Qalb, albumin index; SHR/SP, spontaneously hypertensive stroke prone rats; SIVD, subcortical ischaemic vascular disease; SVD, small vessel disease; TGF-β1, transforming growth factor-β1; TIMPs, tissue inhibitors of metalloproteinases; TNF-α, tumour necrosis factor-α; UCAO, unilateral carotid artery occlusion; VCID, vascular cognitive impairment and dementia; VEGF-1, vascular endothelial growth factor-1; WM, white matter; WMHs, white matter hyperintensities
- © 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society