HIT ONE: Microvascular Circulation

1. Introduction: References

100 years after Alois Alzheimer described the described the pathologic hallmarks of AD, a new hypothesis emerged to explain how the brain gets to that end stage disease. This is the Berislav Zlokovic neurovascular theory, dubbed the "Two-Hit" theory. In the Two Hit theory (shown in diagram above) hit one is vascular damage and hit two is accumulation of amyloid-beta. 

Application of two hit theory works best if late onset AD is considered two different diseases as regards etiology.

In my opinion, AD in APOE4 carriers should be considered a predominantly genetic disease with an age related component; while AD in non-APOE4 carriers I consider a pure age-related disease.

In regard to AD in non-E4 carriers, various age-related factors can contribute to development of AD. These include elevation of amyloid-beta due to age-related decrease in autophagy. However, a number of age-related factors can also contribute to vascular damage so as to cause hit one.

 I consider the Zlokovic two hit theory as a perfect fit for the finding related to etiology and pathogenesis of AD in APOE4 carriers.


These 5 references, which are all "open-access" are recommended for study:

2011, "Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders", Zlokovic.(31)

***2012, "Apolipoprotein E controls cerebrovascular integrity via cyclophilin A" Bell, Winkler, Zlokovic, et.al.(12)

2013, "Cerebrovascular Effects of Apolipoprotein E; Implications for Alzheimer's disease", Zlokovic.(32)

2014, "The pericyte: A Forgotten Cell Type with Important Implications

for Alzheimer's disease ?", Winkler, Sagare, Zlokovic, (33)

***2015, "Accelerated pericyte degeneration and blood-brain barrier breakdown in apolipoprotein E4 carriers with Alzheimer's disease", Halliday, Winkler, Zlokovic, et.al. (13)

Consideration of "hit one" the importance of vascular damage starts with the following observations:

"The importance of the circulatory system to the human brain is highlighted by the fact that although the brain comprises ~ 2% of total body mass, it receives up to 20% of cardiac output and is responsible for ~ 20% and ~25% of the body's oxygen consumption and glucose consumption, respectively. To underline this point, when cerebral blood flow stops, brain function ends within seconds and damage to neurons occurs within minutes." [2011, Zlovokic, above]

The brain circulatory system is 400 miles in length.

AD Etiology: APOE4-- LRP1-- CypA-- NFkB--MMP9--BBB

LRP1 is a very important transport protein in the brain. 

LRP1 combines strongly with APOE2 and APOE3; but combines VERY WEAKLY WITH APOE4. 

The failure of APOE4 to combine properly with LRP1 transport protein is a extremely important lack of function genetic defect.  

It is this specific defect which makes APOE4 carriers at high risk for AD. 

LRP1 failure to properly bind to APOE4 results in failure to properly transport CypA. The rsults in CypA being redistributed from neurons to vascular component. In the vascular area CypA becomes activated and functions as proinflammatory substance. This triggers nuclear factor-kB (NF-kB) which causes increased production of matrix-metalloproteinase-9 (MMP9), The function of MMP9 is to dissolve proteins. The activation of NF-kB--MMP9 pathway results in inflammation leading to loss of pericytes and breakdown of BBB.

APOE4 failure to properly bind to LRP1 also results in decreased transport of AB and accumulation of amyloid-B in interstitial space.

In following section will examine in detail two Zlokovic studies, a 2012 mouse study (12)and a 2015 study of post-mortem human brains(13), which focus on the above pathway and how leads to breakdown of BBB.

Hit One leads to Hit 2

Hit One is the slow track leading to very gradual deterioration of the microcirculation. Hit 2 is the fast track to dementia.

Hit One is the target for prevention.

AD Etiology in APOE4, Mouse Study, Human Post-Mortem Brain

Mouse study: APOE4--LRP1--CypA--MMP9: A Leaky BBB

The main thesis of this website and approach to prevention of AD in APOE4 carriers is that AD in APOE4 carriers is a distinct disease as regards ETIOLOGY from AD in non-E4 carriers. 

This 2012 Zlokovic mouse study combined with the 2015 Zlokovic post- mortem human brain study, (below) more than anything else, proves this thesis.

 "Apolipoprotein E controls cerebrovascular integrity via cyclophilin A",  Bell, Zlokovic, 2012 (12) is a watershed study in the understanding of AD in APOE4 carriers. The study uses transgenic mice in which murine APOE gene is replaced with human APOE4 gene. The study shows how the APOE4 gene leads to break down of microcirculation and BBB.

The specific etiological event is that high levels of CypA (cyclophilin A) causes breakdown of BBB leading to vascular defects.

To study the effects of APOE on brain microcirculation they studied mice with replacement of murine APOE with each human APOE isoform, (E2, E3, E4) plus mice lacking APOE. The focus was on molecules that mediate BBB dysfunction and main focus was CypA. 

[Note: The problem with APOE4 is lack of function regarding doing job of maintaining cerebrovascular integrity; therefore, APOE4 and lacking APOE is functionally the same.]

They initially demonstrated an intact BBB in mice with APOE2 and APOE3 and leaky BBB in APOE4 mice and APOE null mice. 

Next demonstrated that APOE4 mice and APOE null mice had 5-6 fold higher levels of CypA in cerebral microvessels. Data suggested that APOE2 and APOE3 effectively maintain physiologic levels of CypA expression in pericytes. 

Next step demonstrated that CypA deficiency or inhibition reverses BBB breakdown in APOE4 mice and APOE null mice. 

CypA was inhibited by cyclosporine, a drug that binds CypA and inhibits effect. 

After BBB disruption had developed, administration of cyclosporine for 7 days blocked action of CypA and restored integrity of BBB so no longer had leaky BBB. 

Leaky BBB caused accumulation of thrombin, fibrin and haemosiderin (from RBC microbleeds). Thrombin is neurotoxic, fibrin causes neurovascular damage and haemosiderin generates reactive oxygen species, thus implicating multiple potential BBB-derived sources of injury. 

To elucidate mechanism of injury showed that CypA activates NF-kB (nuclear-factor-kB) which activates MMP9. The result is activated MMP9 acts on collagen IV and tight-junction proteins to degrade capillary basement membrane and disrupt BBB. 

In the mice, the chronic breakdown of BBB caused microvascular reductions which included: 

Microvascular degeneration,

DNA fragmentation in endothelial cells and pericytes,

Diminished pericyte coverage of basement membrane,

Reductions in microvascular length.

This all correlated with reduction of regional cerebral blood flow.

All these deteriorations to BBB and microvascular system were blocked by blocking CypA.

They then demonstrated that accumulation of CypA is directly related to LRP1 (lipoprotein receptor related protein 1) as follows:


APOE3 binds firmly to LRP1.

APOE4 does not bind to LRP1.

 LRP1 was required for APOE3 inhibition of CypA.

When eliminated LRP1, APOE3 could not inhibit CypA and leaky BBB developed.

They demonstrated that because APOE4 does not bind to LRP1, APOE4 is unable to properly regulate physiologic CypA levels in cerebral microvessels which then results in breakdown of BBB.

They also demonstrated that vascular defects in APOE4 mice and APOE null mice develop before neuronal dysfunction. Vascular defects are present at 2 weeks of age; but nerve function is normal at that age. At 4 months vascular defects have progressed and nerve defects appear. 

The nerve defects can then be ameliorated by giving Cyclosporine which normalizes the BBB and then nerve deficit improve.

The paper concludes: "Understanding the contribution APOE4 to the pathogenesis of Alzheimer's disease may be one of the most important avenues to a new therapy...Our finding demonstrate that APOE maintains cerebrovascular integrity necessary for normal neuronal function by regulating the CypA--NF-kB--MMP9 pathway in pericytes in an isoform-specific manner. 

"We also show that CypA is a key target for treating APOE4-mediated neurovascular defects and the resulting neuronal dysfunction."

What makes this 2012 mouse study of extraordinary importance is the following 2015 Zlokovic study of post-mortem brains of persons with AD, both APOE4 and APOE3. 

The same results are shown in human ADOE4 AD brains. Human AD brains in APOE4 carriers have marked breakdown of BBB and increase in CypA and MMP9 in pericytes and endothelial cells plus loss of pericytes, diminished pericyte coverage of basement membrane and reduction in microvascular length.

Post-Mortem studies in Human Brain in AD: APOE4 vs APOE3

This extremely important paper is study of post-mortem finding of human brains in age matched controls of APOE3 persons who died of non-neurologic disease versus APOE3 persons who died of AD versus APOE4 persons who died of AD. The three groups are compared as to specific findings at autopsy.

The paper is "Accelerated pericyte degeneration and blood-brain barrier breakdown in apolipoprotein E4 carriers with Alzheimer's disease, Halliday, Zlokovic, 2015. (13) It is a study about what is different in the AD brain on APOE4 carriers versus APOE3. It is somewhat limited as shows end stage disease; so early pathogenesis must be inferred from late finding; but still very revealing. The finding are summarized below. Finding are APOE controls compared to AD brains in APOE3 and the AD APOE3 brains compared to APOE4 brains.

Autopsy finding in Human Brain

                                            APOE3, controls vs            APOE3 AD vs            APOE4 AD brains

Number Pericytes                                               --30%                          --31% 

Pericyte coverage                                               --23%                          --15% 

Capillary length                                                   --14%                          --20%

BBB function

 Fibrin                                                               6.9 fold increase            3.1 fold increase 

IgG                                                                    5.3 fold increase            2.6 fold increase 

CypA pericytes                                                3.4 fold increase             46% increase 

CypA endothelial cells                                   1.4 fold increase             70% increase

MMP9 pericytes                                              32% increase                   82% increase 

MMP9 endothelial cells                                 79% increase                  132% increase 

The autopsy finding showed the brains of APOE3 AD patients had 30% fewer remaining pericytes than the age matched APOE3 controls without AD and 23% less basement membranes coverage. The E4 AD patients had 31% fewer pericytes and 15% less pericyte coverage of basement membrane than E3 AD patients. Decreased pericytes and pericyte coverage of basement membranes was associated with 14% reduction in capillary length of E3 controls vs E3 AD patients and 20% reduction in capillary length in AD E4 patients vs E3 AD patients. 

There was a breakdown in BBB integrity in AD E3 patients compared to E3 controls with 6.9 fold increase in fibrin and 5.3 fold increase in IgG in brain. The E4 AD patients showed a 3.1 fold increase in fibrin and a 2.6 fold increase in IgG in brain compared to E3 AD patients.

The E3 AD patients showed a increase in CypA in pericytes and endothelial cells of 3.4 fold and 1.4 fold,, respectively compared to E3 controls. Likewise the E4 AD patients had a 46% and 70% increase in CypA compared to E3 AD patients. 

The increase in MMP9 showed a 32% and 79% increase in E3 AD pateints compared to controls. The E4 AD patients showed a 82% and 132% increase compared to E3 AD patients.

The E3 AD patients and the E4 AD patients showed an equal reduction of LRP1 levels compared to controls. The reduction was 55% in pericytes and 74% in endothelial cells.

"A substantial reduction in LRP1 endothelial levels found in this study is consistent with a previous report of 75% reduction in LRP1 brain endothelial level in AD patients compared with non-AD controls." (13)

[Note: It is this reduction in LRP1 with AGE in Non-E4 persons which could be  basis of age-related deterioration of cerebral vascular system and BBB in aged persons without E4 allele.]


"Recent studies in murine transgenic models have shown that pericytes have a critical role in maintaining the BBB integrity and that loss of pericytes can lead to long-term BBB breakdown and small vessel disease contributing to neurodegenerative changes."

Pericytes degenerate in AD. Multiple studies have demonstrated loss of cerebrovascular integrity and/or BBB damage in AD that is associated the APOE4 genotype...Individuals with one copy of APOE4 have a 3.7-fold increase in AD risk and individuals with two copies of APOE4 a 12-fold increase in AD risk relative to APOE2/APOE3 individuals. Studies using murine transgenic models have show that APOE4 increases BBB susceptibility to injury and leads to BBB breakdown and microvascular reductions in humanized transgenic APOE4 mice compared to APOE3 mice."

"Recent studies in transgenic APOE2 and APOE3 mice have shown that astrocyte-secreted apoE2 and apoE3 maintain BBB integrity by suppressing the proinflammatory CypA-MMP9 pathway in pericytes via low-density lipoprotein receptor-related protein-1 (LRP-1) that is a major apoE receptor. In contrast, astrocyte-secreted apoE4 fails to effectively suppress CypA--MMP9-pathway in APOE4 transgenic mice leading to MMP9-mediated degradation of BBB tight junction and basement membrane proteins, which cause BBB breakdown. (Ref 2, 2012 considered above).

"Using postmortem human brain tissue analysis of biomarkers of BBB breakdown, here we show that APOE4 compared with APOE3 accelerates pericyte loss and microvascular reductions in AD, which correlates with the magnitude of BBB breakdown to plasma proteins immunoglobulin G and fibrin. We then show that APOE4 compared to APOE3 leads to greater accumulation of CypA and MMP-9 in pericytes and endothelial cells in AD suggestive of an enhance activation of LRP1-dependent CypA--MMP9 BBB degrading pathway, which in addition to pericyte loss may contribute to accelerated BBB breakdown in AD APOE4 compared to AD APOE3 carriers."



"In this study we show that AD APOE4 carriers develop accelerated pericyte degeneration and BBB breakdown compared with AD APOE3 carriers, whereas AD APOE3 carriers show a significantly greater loss of pericytes compared with non-AD controls, which correlate with the magnitude of BBB breakdown to the studied plasma proteins fibrin and IgG...The present study (shows that) a greater BBB disruption (is related to) greater loss of pericyte population that has been shown to lead to small vessel disease and chronic BBB breakdown in murine transgenic models and is associated with BBB breakdown" in AD.

"At the molecular level, we show increased accumulation of CypA and MMP-9 in pericytes and endothelial cells in AD APOE4 carriers compared with AD APOE3 carriers, which, in turn, develop greater levels of CypA and MMP-9 in the vascular cells compared with non-AD controls. In APOE4 transgenic mice, activation of CypA-MMP-9 pathway in pericytes has been shown to mediate BBB breakdown by degrading BBB tight-junction proteins,,, and the basement membrane proteins of the capillary wall, collagen IV that are all substrates for MMP-9. 

ApoE3, but not ApoE4, effectively inhibits CypA-MMP-9 pathways in pericytes in vitro and in transgenic mice in vivo acting through LRP1, which leads to LRP1- dependent transcriptional suppression of CypA and a subsequent transcriptional MMP-9 inhibition, as reported. Compared with ApoE3, ApoE4 interacts weakly with LRP1 on vascular cells, which leads to a loss of inhibition of CypA-MMP-9 pathway and its activation over time causes a progressive age-dependent BBB breakdown as shown in transgenic APOE4 mice."

"LRP1 levels are reduced in brain capillaries in AD that is not influenced by APOE genotype"

"This likely contributes to BBB breakdown in AD APOE3 similar to that reported in transgenic APOE3 mice after LRP1 silencing. "... The degree of BBB breakdown in AD APOE3 carriers was...less pronounced than in APOE4 carriers." ApoE4 is a much weaker ligand for LRP1 in vascular cell compared to apoE3 and is much less effective in inhibiting elevating CypA--MMP-9 pathway in human AD. Thus APOE4 have a double hit, weak ligand-receptor affinity plus reduced levels while AD APOE3 only have single hit, reduced LRP1 levels. 

"Interestingly, we also found that loss of pericyte coverage, extravascular accumulation of plasma derived proteins (i.e., IgG) and increased CypA levels,..in pericytes correlate with Braak stage and cognitive impairment. These data may suggest a possible role of BBB breakdown in the development of neuropathology and dementia in AD."

"The present study suggests BBB breakdown in AD APOE4 carriers could be related to accelerated pericyte degeneration and/or activation of CypA--MMP-9 pathway...It is possible that activation of MMP-9 accelerates pericyte loss by at least two pathways:

(1) by allowing a greater ingress of blood-derived products across the BBB that are taken up and degraded by pericytes, which, over time, has been shown to lead to pericyte loss in animal models, and

(2) by degrading the extracellular matrix and important cell adhesion molecules around brain capillaries that can lead to separation of pericytes from the capillary wall and their cell death." 

In my opinion, the above data shows the following:

AD in APOE4 carriers is different from AD in non-E4 carriers in that APOE4 carriers have greater loss of pericytes, capillary coverage and capillary length. Greater breakdown of BBB. Greater increase in CypA and MMP9 in pericytes and endothelial cells.

The study combined with the 2012 Zlokovic mouse study above, shows that AD in APOE4 carriers has a different etiology than AD in non-APOE4 carriers. The specific etiology is the failure of ApoE4 to form a strong bond with LRP1. This loss of function defect results in increase activity of CypA and MMP9 which results in loss of pericytes, pericyte coverage, capillary length all leading to breakdown of BBB.

Note: This is same pathology saw in 2015 study in mice with human APOE4 genes and damage blocked by Rapamycin.