Cerebrovascular Endothelial Dysfunction: Role of BACE1

Abstract

Dysfunctional endothelium is increasingly recognized as mechanistic link between cardiovascular risk factors and dementia, including Alzheimer’s disease (AD). Beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is responsible for beta-processing of APP, the first step in production of amyloid beta (Aβ) peptides, major culprits in pathogenesis of AD. Under pathological conditions, excessive activation of BACE1 exerts detrimental effects on endothelial function by Aβ-dependent and Aβ-independent mechanism. High local concentration of Aβ in the brain blood vessels is responsible for the loss of key vascular protective functions of endothelial cells. More recent studies recognized significant contribution of Aβ-independent proteolytic activity of endothelial BACE1 to pathogenesis of endothelial dysfunction. This review critically evaluates existing evidence supporting the concept that excessive activation of BACE1 expressed in cerebrovascular endothelium, impairs key homeostatic functions of the brain blood vessels. This concept has important therapeutic implications. Indeed, improved understanding of the mechanisms of endothelial dysfunction may help in efforts to develop new approaches to protection and preservation of healthy cerebrovascular function.

Graphical Abstract

1. Introduction

In brain, endothelial cells play critically important roles in preservation of vascular and neuronal function.^1,2,3 They significantly contribute to control of vasomotor function, neurovascular coupling, and protection of biochemical, structural, and functional integrity of the central nervous system. Endothelium produces substances that could be released into luminal or abluminal space of cerebral blood vessels⁴ thus affecting function of circulating cells, perivascular cells, and brain parenchymal cells.^1,4 In this review, we focus on the role of endothelial β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) in the pathogenesis of cerebrovascular endothelial dysfunction. We address contributions of amyloid-dependent and amyloid-independent proteolytic activity of BACE1.

2. Endothelial dysfunction in human cerebral blood vessels

Efforts to define the role of endothelium in the pathogenesis of cardiovascular disease demonstrated that reduced production, release, and biological activity of endothelial NO are key mechanisms in development of endothelial dysfunction.^5,6,7 Loss of NO is responsible for vasoconstrictor, pro-inflammatory, and pro-thrombotic phenotype of dysfunctional endothelium.^5,6,7 In contrast, genetic predisposition to enhanced endothelial NO signaling in humans is associated with reduced risks of stroke, heart disease, and peripheral arterial disease.⁸ Endothelium becomes dysfunctional in blood vessels chronically exposed to cardiovascular risk factors including hypertension, diabetes, hypercholesterolemia, and aging.^5,6 Existing literature suggests that endothelial dysfunction is responsible for initiation and progression of atherosclerosis, and it is considered hallmark and predictor of cardiovascular disease.⁵ More recent evidence suggests that loss of endothelial NO function in the brain, is a molecular mechanism linking endothelial dysfunction with development of cognitive impairment.^9,10 Indeed, loss of endothelial NO upregulates expression of BACE1, promotes amyloidogenic processing of APP, phosphorylation of tau protein, leakage of blood-brain-barrier (BBB), activation of microglia, microvascular occlusion, and damage of white matter.^11–14 Relevance of cerebrovascular endothelium in preservation of normal cognitive function is further underscored by reports demonstrating that endothelial dysfunction is common mechanism in pathogenesis of cerebral small vessel disease (cSVD), neurodegenerative pathology, and cognitive impairment.^15–18

Early studies of endothelial dysfunction in humans were performed by analysis of endothelium-dependent vasodilatation in forearm circulation and coronary arteries.^19–21 Impaired endothelium-dependent vasodilator responses served as surrogate endpoint for detection of endothelial dysfunction. Since in vivo methodologies used in analysis of vasomotor function in systemic blood vessels are not applicable to the human cerebral circulation, it has not been possible to directly examine cerebrovascular endothelium-dependent vasodilatation in response to acetylcholine or flow-induced vasodilatation in the human brain. Nonetheless, it has been shown that pharmacological inhibition of NO synthesis reduces cerebral blood flow in healthy subjects.^22,23 Furthermore, impairment of endothelium-dependent vasodilatation in systemic arteries correlates with white matter hyperintensities,²⁴ and endothelial dysfunction in brachial artery is associated with the severity of cSVD.²⁵ These observations imply that impairment of endothelium-dependent vasodilatation is also present in the human cerebral blood vessels. Consistent with this interpretation, postmortem analysis of vasomotor function of the brain arterioles derived from patients with cSVD demonstrated impaired endothelium-dependent relaxations to acetylcholine.²⁶ Positive results of recently completed LACI-2 clinical trial designed to evaluate “endothelial-stabilizing drugs”, isosorbide mononitrate and cilostazol, in patients with cSVD and lacunar stroke, reinforced the conclusion that dysfunctional endothelium is major therapeutic target in prevention of cSVD and cognitive impairment.²⁷ We also wish to point out that beneficial effects of cilostazol could be explained in part by stimulatory effect of cilostazol on clearance of amyloid beta (Aβ) as reported by recently completed Cilostazol for Prevention of Conversion from Mild Cognitive impairment to Dementia (COMCID) clinical trial.²⁸

3. BACE1

3.1. Function of BACE1

BACE1 is type I transmembrane protein widely expressed in the brain neurons, oligodendrocytes, and astrocytes.²⁹ Sequential cleavage of APP by BACE1 and gamma (γ)-secretase is responsible for production of amyloid beta (Aβ) peptides (Aβ40 and Aβ42; Figure 1). According to amyloid hypothesis, deposition and accumulation of Aβ peptides in the brain is considered major mechanism in the pathogenesis of AD.³⁰ BACE1 is a member of evolutionary conserved large superfamily of aspartic proteases.³¹ The fact that the evolutionary origin of BACE1 and γ-secretase is much more ancient than that of APP suggests that these enzymes participate in control of additional and diverse fundamental cellular functions independent of their role in APP processing.³¹ Indeed, about 70 BACE1 substrates have been identified.²⁹ The levels of BACE1 mRNA are highest in brain and pancreas.²⁹ Out of six alternatively spliced isoforms (A-F), BACE1 isoform A exhibits the highest protease activity.²⁹ Physiological roles of BACE1 in the central nervous system include regulation of myelination, axon guidance, synaptic function, and neurotransmission (reviewed in Hampel et al., 2021).³² BACE1 mRNA and protein are also expressed in pancreatic-β cells, adipocytes, hepatocytes, and vascular endothelial and smooth muscle cells²⁹. Expression of BACE1 has been detected in endothelium of cerebral blood vessels yet the exact vascular functions of BACE1 remain to be determined.^11,33–35

Figure 1.

In early stages of cerebrovascular endothelial dysfunction loss of endothelial nitric oxide synthase (eNOS) function causes pathologic elevation of abluminal proteolytic cleavage of amyloid precursor protein (APP) by β-secretase (β) and γ-secretase (γ) thereby resulting in generation of soluble APPβ (sAPPβ) and Aβ. Aβ activates CD36 receptors on border associated macrophages (BAM) and stimulates production of superoxide anion (.O2-). Chemical reaction between .O2- and nitric oxide (NO) reduces local concentration of NO and generates a potent oxidant, peroxynitrite (ONOO-). Oxidation of tetrahydrobiopterin (BH4) by ONOO- causes uncoupling of eNOS. Loss of eNOS function exacerbates upregulation of BACE1 and increased production of Aβ40 and Aβ42 thus propagating vicious cycle. C99 = 99-aa C-terminal fragment of APP, AICD = APP-intracellular domain of APP.

3.2. Regulation of BACE1 gene expression in cerebrovascular endothelium

BACE1 gene expression in neuronal and glial cells is regulated by multiple mechanisms involving complex interaction between activators and inhibitors of BACE1 transcription.³⁶ Transcription factors specificity protein 1 (Sp1), yin yang 1 (YY1), and nuclear factor-κB (NF-κB) exert stimulatory effect on BACE1 transcription whereas peroxisome proliferator-activated receptor-γ (PPAR-γ) serves as repressor of BACE1 transcription.³⁶ Few reports are available in current literature regrading control of BACE1 expression in cerebrovascular endothelium. Studies on cultured human brain microvascular endothelial cells and in vivo studies on murine cerebral microvessels established that endothelial NO exerts inhibitory effect on expression of BACE1.¹¹ This effect is mediated by NO-induced activation of soluble guanylate cyclase and increased production of cyclic GMP.¹¹ While the exact downstream signaling by NO/cyclic GMP in cerebrovascular endothelium remains to be determined, exiting evidence suggests that PPAR-γ-coactivator 1-α (PGC-1α) is major mediator of the effects of NO/cyclic GMP.³⁷ Moreover, similar mechanism of NO/cyclic GMP/PGC-1α signaling appears to be responsible for the inhibitory effect of NO on expression of BACE1 in neuronal cells.³⁸

We also wish to point out that hypoxia is strong stimulator of BACE1 expression in cerebrovascular endothelium (see section 4.3.). This effect is mediated by hypoxia-inducible factor 1-α.^39,40 In addition, treatment with hydrogen peroxide increases expression of BACE1 protein in cultured human microvascular endothelial cells.⁴¹ This effect is prevented by cystatin C-induced degradation of BACE1 mediated by activation of the ubiquitin/proteasome pathway.⁴¹ Furthermore, treatment with astaxanthin (a xanthophyll carotenoid nutraceutical), reduced BACE1 mRNA expression levels in murine brain capillary endothelial cells derived from 3xTg AD mice.⁴² Of note, astaxanthin can prevent oxidative stress and cognitive deficits in murine models of Alzheimer’s disease by activation of the SIRT1/PGC-1α signaling pathway.⁴³ Whether same signaling pathway may be activated by astaxanthin in the brain endothelial cells of patients with AD is currently unknown.

4. Amyloidogenic function of BACE1

4.1. BACE1 and Antimicrobial Protection Hypothesis of AD

For many years, Aβ peptides have been considered byproducts of APP catabolism lacking vascular or neuronal function. However, the human Aβ sequence is highly conserved across most vertebrate species.⁴⁴ Consistent with 400 million years of evolutionary conservation, accumulating evidence continues to substantiate functional importance of Aβ peptides.⁴⁴ While physiological role of Aβ in endothelium is still incompletely understood, recent findings suggest that Aβ may function as antimicrobial peptide.^44,45 Antimicrobial peptides (AMPs) consist of 12–50 amino acid residues with antimicrobial activity, and they are vital component of innate immunity.⁴⁶ AMP role of Aβ produced in endothelium is in agreement with recognized contribution of endothelial cells to innate immunity.^46–48 Indeed, endothelial cells have functions traditionally assigned to immune cells including phagocytosis, cytokine/chemokine secretion, and antigen presentation.^49–51 In addition, endothelial cells in the brain constituting blood-brain-barrier (BBB) produce AMP, cathelicidin LL-37, thus suggesting that BBB has bactericidal properties designed to protect brain from circulating pathogens.⁵²

The recognition of AMP function of Aβ provided foundation for development of antimicrobial protection hypothesis of AD.⁴⁴ According to this hypothesis, dysregulated response of immune system to increased microbial burden exacerbates production and deposition of Aβ deposition in the cerebral blood vessels and brain parenchyma.⁴⁴ Thus, excessive production and release of Aβ from cerebrovascular endothelium, may provide underappreciated contribution to development of cerebral amyloid pathology.^14,31,33 It is also important to note that depending on local concentration, cellular environment, and characteristics of cell membrane, Aβ exerts cytotoxicity comparable to cytotoxic effect of AMPs.⁴⁴ Both Aβ and AMPs have dual protective/damaging roles.⁴⁷ The cytotoxicity of Aβ exerts wide range of detrimental effects on cerebrovascular endothelial function including deterioration of endothelial mitochondrial function, impairment of BBB permeability, and endothelial cell death.^53,54

4.2. Endothelial BACE1 in Alzheimer’s Disease and Cerebral Amyloid Angiopathy

We wish to emphasize that endothelial dysfunction is an early event in the development of Alzheimer’s disease (AD).^55,56 Ongoing therapeutic efforts have been focused on early detection and treatment during preclinical stages of AD. In this regard, existing literature favors prevention of endothelial dysfunction as important component of overall approach to protection and preservation of healthy cognition. Endothelial dysfunction mediated by the loss of endothelial NO stimulates expression of BACE1 and APP protein as well as production of Aβ, which in turn, further exacerbates loss of NO by Aβ-induced activation of CD36 on border associated macrophages and subsequent increase in production of reactive oxygen species.⁵⁷ Reaction between superoxide anion and NO lowers concentration of NO and generates peroxynitrite, a very potent oxidant.⁵⁸ Oxidation of tetrahydrobiopterin (BH4, an essential cofactor required for eNOS enzymatic activity and production of NO) by peroxynitrite causes uncoupling of eNOS and further exacerbates endothelial dysfunction.⁵⁹ Perpetuation of created eNOS/BACE1/Aβ vicious cycle may cause severe dysfunction of neurovascular unit and deposition of Aβ peptides in the brain blood vessels and neuronal tissue.^60,61 In agreement with this hypothesis, eNOS^−/− and eNOS^+/− mice suffer from CAA and AD pathology as well as learning and memory impairments.^9,61 Structural alterations detected in the brains of eNOS^−/− mice provide an additional explanation for observed behavioral impairments.⁶²

Of particular interest to this review, more recent studies designed to define cellular localization of BACE1 in cerebrovascular endothelium indicate that in BBB, BACE1 is predominantly localized in abluminal cell membrane. Indeed, abluminal levels of BACE1 protein are ~2.5 higher as compared to levels detected in luminal membrane of endothelial cells³⁴ (Figure 1). Moreover, in mouse model of AD, significant upregulation of BACE1 in BBB endothelial cells have been detected. Given abluminal localization of BACE1, it appears that excessive endothelial production of Aβ, may contribute to deposition and accumulation of Aβ in the brain blood vessels thereby instigating and enhancing development of CAA.³⁴ These observations suggest that drugs selectively targeting brain endothelial BACE1 might offer viable therapeutic approach in prevention of CAA.³⁴ Of note, BACE2, close homologue of BACE1, is also expressed in the brain endothelial cells, however, BACE2 exerts endothelial protective effects thereby guarding cerebral vasculature from CAA.⁶³

We also would like to point out that findings obtained in old (18 month of age) eNOS-heterozygous (eNOS^+/−) knockout mice suggest that significantly higher levels of Aβ are produced and released from cerebral microvessels of eNOS^+/− as compared to Aβ levels released from cerebral microvessels of aged matched wild type mice.⁶⁰ Notably, this increase of Aβ is detectable before any changes of Aβ levels in the brain parenchyma. These observations provide additional support for possible contribution of endothelial BACE1 to early stages of excessive amyloid production in cerebral blood vessels suffering from partial loss of eNOS function.⁶⁰

4.3. Hypoxia

Upregulation of APP expression and enzyme activity of BACE1 and γ-secretase in response to hypoxia are strongly conserved during evolution.³¹ These ancient responses of brain to hypoxia are present in zebrafish.⁶⁴ Exposure of cultured endothelial cells to hypoxia promotes β-processing of APP and results in increased production of Aβ.^39,40,65 Mechanistically, hypoxia-induced factor-1α is responsible for transcriptional activation of BACE1 and increased β-processing of APP under hypoxic conditions. Since hypoxia is strong stimulus for angiogenesis, prior studies have focused on contribution of Aβ to formation of new blood vessels in the brain. However, the role of Aβ in angiogenesis remains controversial.⁶⁶ Furthermore, in brains derived from patients with AD, aberrant angiogenesis is manifested by formation of “string vessels.” These non-functional capillaries are mostly composed of connective tissue and devoid of endothelial cells.^67,68 In some cases, formation of string vessels may also promote phenomenon of “vascular bagging” associated with disruption of the blood brain barrier and accumulation of the plasma proteins in vascular bags.⁶⁸

Obstructive sleep apnea (OSA) is condition associated with hypoxia-induced upregulation of BACE1 and subsequent cognitive impairment.⁶⁹ Upregulation of BACE1 expression and increased β-processing of APP has been detected in brains of rats exposed to chronic intermittent hypoxia.⁷⁰ Moreover, AD biomarkers are elevated in patients with OSA.^71,72 The in vivo effect of intermittent hypoxia or OSA on expression of BACE1 in human cerebral blood vessels have not been studied. Given strong evolutionary conservation of increased β-processing of APP in response to hypoxia, as well as the results obtained in hypoxic cultured human endothelia cells, it is very likely that OSA is associated with endothelial dysfunction mediated in part by upregulation of endothelial BACE1. Same mechanism may help explain why ischemic/hypoxic conditions including stroke, cerebral small vessel disease, and heart arrest, may also increase the odds of developing AD.⁷³

4.4. Hypertension

Prior studies established that in murine model of AD, hypertension promotes β-processing of APP and enhances Aβ-induced cerebrovascular endothelial dysfunction.⁷⁴ Moreover, even though human hypertension is not considered a risk factor for development of CAA,⁷⁵ hypertension also increases microvascular amyloid deposition in Tg2576 mice.⁷⁴ More recent study demonstrated that in mice, overexpression of BACE1 in endothelial cells increases circulating levels of Aβ1–40 and Aβ1–42⁷⁶ and that elevated circulating levels of Aβ peptides were associated with reduced cortical cerebral blood flow. Mechanistically, reduced cerebral blood flow was ascribed to the inhibitory effect of circulating Aβ peptides on eNOS activity mediated by aberrant phosphorylation of eNOS protein.^76–78 In addition, endothelial BACE1 exerts inhibitory effect on eNOS by increased association of eNOS with caveolin-1, and by reduced expression of glypican-1 (GPC-1)^76,79 (these findings will be discussed in the next section on amyloid-independent effects of BACE1). We wish to emphasize that significantly higher BACE1 protein expression has been detected in cerebral microvascular endothelial cells derived from patients with hypertension, and hypertensive patients with cSVD.⁷⁶ The exact contribution of excessive β-processing of APP in the human cerebrovascular endothelium to hypertension-induced cognitive impairment is incompletely understood and remains to be investigated.

Of particular interest to this review, global genetic inactivation of BACE1 (in BACE1^−/− mice) protects against hypertension induced by high fat diet.⁸⁰ Since genetic inactivation of BACE1 is also associated with lowering of blood glucose, and body weight,⁸¹ these systemic effects may contribute to overall antihypertensive effect of BACE1 inactivation. However, intravenous infusion of murine Aβ42 results in increased arterial blood pressure in control mice and mice fed high fat diet.⁸⁰ These are important observations demonstrating that increased circulating concentrations Aβ42 may exert significant prohypertensive effect mediated in part by detrimental effects on endothelial and smooth muscle function.

4.5. Diabetes

The effect of diabetes on expression and function of BACE1 in human cerebrovascular endothelium has not been studied. However, recent findings suggest that circulating concentration of Aβ42 is significantly increased (no change was detected in circulating concentration of Aβ40) in mouse model of metabolic stress (obesity and diabetes) and in patients with type 2 diabetes.⁸⁰ Most importantly, increased plasma levels of Aβ42 appear to be sufficient to impair endothelium-dependent relaxations to acetylcholine, reduce activity of eNOS, and inhibit production of cyclic GMP in microvessels of mouse skin.⁸⁰ Interestingly, elevated concentrations of Aβ42 are also able to impair reactivity of smooth muscle cells to NO thus suggesting that aberrant function of smooth muscle cells contribute to overall impairment of endothelium-dependent vasodilatation. In patients with type 2 diabetes, impairment of endothelium-dependent vasodilatation is also associated with elevated circulating concentration of Aβ42 thereby suggesting that endothelial dysfunction detected in diabetic mice might be recapitulated in patients with diabetes.⁸⁰ It is however important to keep in mind that dysfunction of endothelial and smooth muscle cells caused by Aβ42 were detected in the systemic blood vessels, and that these findings may not accurately predict phenotypic alterations induced by Aβ42 in the brain vasculature of diabetic patients. Moreover, potential contribution of the BACE1 expressed in cerebrovascular endothelium to Aβ-mediated detrimental effects of diabetes on the brain circulation remains to be defined.

Binding of circulating insulin to insulin receptor (IR) on endothelial cells activates protein kinase B (Akt) thus leading to phosphorylation of eNOS on stimulatory site Ser1177 and increased production of NO.^81,82 In contrast, in vivo endothelium-specific inactivation of IR results in reduced expression of eNOS and endothelial dysfunction.⁸³ These observations show that intact insulin receptors are critically important in preservation of normal endothelial function.⁸⁴ Relevant to our review, existing evidence support the concept that in liver, increased activity of BACE1 impairs insulin signaling by proteolytic cleavage of IR⁹⁶ (see Section 5.3) or by excessive production of Aβ.²⁹ Elevated local concentrations of Aβ directly compete with insulin for binding to IR⁸⁵ thus disrupting insulin signaling. We speculate that if operational in the cerebrovascular endothelium, these inhibitory effects of BACE1 on IR signaling may exert detrimental effects on eNOS functions and insulin-dependent mechanisms responsible for preservation of intact BBB.⁸⁶

5. Amyloid-independent effects of BACE1

5.1. Hypertension

Brain microvascular endothelial cells of hypertensive patients and hypertensive patients with cSVD have significant increase in expression and enzymatic activity of BACE1 in cerebrovascular endothelium⁷⁶ (Figure 2). Most importantly, elevated activity of BACE1 in endothelium causes proteolytic degradation of tight junction protein occludin.⁷⁶ In addition, levels of other tight junction proteins, zonula occludens-1 (ZO-1), junctional adhesion molecule-A (JAM-A), and claudin-1 are also decreased even though unlike occludin, these proteins are not affected by proteolytic activity of BACE1. The exact mechanisms underlying loss of ZO-1, JAM-A, and claudin-1 remain to be defined. Loss of occludin, as well as ZO-1, JAM-A, and claudin-1 leads to disruption of endothelial tight junctions and increased permeability of BBB.⁷⁶ Identification of occludin as substrate for proteolytic cleavage by BACE1 provides entirely novel mechanistic explanation for previously reported association between upregulation of BACE1 expression and downregulation of occludin in cerebral blood vessels isolated from patients with CAA.³³ Of note, association between eNOS and caveolin-1 is significantly increased following BACE-1 mediated cleavage of occludin thereby exerting additional inhibitory effect on eNOS activity.⁷⁶ Discovery of non-amyloidogenic proteolytic activity of BACE1 responsible for disruption of BBB further supports the concept that endothelial dysfunction in cerebral microvessels is an important contributor to pathogenesis of cSVD, and plausible molecular mechanism linking hypertension-induced endothelial dysfunction with pathogenesis of cognitive impairment.^76,87

Figure 2.

In cerebral blood vessels, hypertension and COVID-19 induce elevation of BACE1 expression causing proteolytic cleavage of occludin thereby disrupting blood-brain barrier (BBB). In addition, hypertension-induced elevation of BACE1 inhibits endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO). Aberrant eNOS/NO signaling causes impairment of vascular protective functions of NO thereby leading to endothelial dysfunction.

Endothelium-specific genetic inactivation of BACE1 in human and murine cerebrovascular endothelium causes significant upregulation of endothelial nitric oxide synthase (eNOS) expression and production of NO.⁷⁹ Analysis of molecular mechanisms linking BACE1 activity with expression of eNOS revealed that inhibition of BACE1 also increases expression of GPC-1 (Figure 2). In agreement with these findings, it has been demonstrated that BACE1 is negative regulator of GPC-1-like protein in the brain of zebra fish.⁸⁸ Prior studies established that GPC-1 mediates stimulatory effect of shear stress on eNOS activity.^89–92 Thus, besides protection of BBB, inhibition of BACE1 may also enhance vascular protective functions of endothelial GPC-1/NO signaling. Of note, GPC-1 is significantly downregulated in retina of spontaneously hypertensive rats.⁹³ Whether this phenomenon is dependent on elevation of BACE1 activity in retinal endothelial cells is unknown.

5.2. Atherosclerosis

Studies in cultured human endothelial cells (E.A.hy926) indicate that inflammatory cytokine tumor necrosis factor-α (TNF-α) causes disruption of endothelial tight junctions and promotes adhesion of monocytes to endothelial cells by increasing expression of BACE1.⁹⁴ This pro-atherosclerotic effect of TNF-α appears to be mediated by proteolytic cleavage of α−2, 6-sialic acid transferase 1 (ST6Gal-I) by endothelial BACE1 and subsequent downregulation of vascular endothelial (VE)-cadherin α−2, 6-sialylation.⁹⁴ Indeed, VE-cadherin is critically important in control of vascular permeability and leukocytes extravasation.⁹⁵ Aberrant α−2, 6 sialylations of endothelial proteins have been proposed as significant mechanism responsible for initiation and progression of atherosclerosis.⁹⁴ However, it is important to note that relevance of this mechanism for pathogenesis of atherosclerosis in vivo is unknown. Future studies will have to determine whether impaired α−2, 6 sialylations of endothelial proteins may contribute to the pathogenesis of cerebrovascular endothelial dysfunction.

5.3. Diabetes

As we have already noted, endothelial IR signaling is an important regulator of expression and function of eNOS.^83,84 Of particular interest to this review, increased levels of glucose stimulate cleavage of IR ectodomain by BACE1 thereby reducing number of structurally intact insulin receptors in the liver.⁹⁶ Additionally, in diabetic patients, the released soluble IR can bind insulin and further exacerbate impairment of insulin signaling.⁹⁷ However, shedding of soluble IR from endothelial cells of diabetic patients have not been reported. The hypothesis that proteolytic cleavage of endothelial IR by BACE1 could be a mechanism contributing to BACE1-induced endothelial dysfunction in cerebral blood vessels remains to be tested. Further studies of this mechanism may also provide new insights into the BACE1/IR-dependent mechanisms participating in control of blood-brain barrier (BBB) function.⁸⁶

5.4. Coronavirus disease of 2019 (COVID-19)

Endothelial cells play a key role in pathogenesis of severe acute respiratory syndrome (SARS) as well as cardiovascular and cerebrovascular complications of infections with coronavirus 2 (Cov-2). Several mechanisms of endothelial dysfunction induced by COVID-19 have been studied and reviewed in current literature.^98–100 We would like to focus on the mechanism underlying contribution of BACE1 to endothelial dysfunction in the brain. Upregulation of BACE1 protein expression was detected in human brain microvascular endothelial cells exposed to SARS-Cov-2 spike S1 subunit protein.¹⁰¹ Consistent with previously reported findings expression of proteins essential for BBB function, occludin, ZO-1, and claudins,⁷⁶ are significantly decreased thereby increasing permeability of BBB. Importantly, leakage of BBB is prevented by treatment with BACE1 inhibitor. Another notable observation is that SARS-Cov-2 spike protein accelerates senescence of the brain endothelial cells. In agreement with this observation, previous studies demonstrated that BACE1 was upregulated in senescent human brain microvascular endothelial cells.¹⁰² SARS-Cov-2 spike protein-induced senescence is also prevented by inhibition of BACE1.¹⁰¹ The exact mechanism of BACE1 upregulation in response to SARS-Cov-2 spike protein has not been defined, however, presented findings support the concept that high expression of BACE1 is an important contributor to development of endothelial dysfunction including disruption of BBB. It is conceivable that impaired permeability of BBB may contribute to cognitive impairment induced by COVID-19.^103–105 Indeed, recent findings demonstrated that BBB is disrupted in patients with long COVID-19 associated with brain fog.¹⁰⁶

6. Therapeutic implications

The scientific rationale for therapeutic application of BACE1 inhibitors in prevention of AD is based on current understanding of critical role of BACE1 in proteolytic cleavage of APP and resulting generation of monomeric forms as well as cytotoxic aggregates of Aβ peptides.³² However, clinical trials with BACE1 inhibitors were discontinued for either futility or safety reasons.³² Major challenge in therapeutic application of BACE1 inhibitors is how to achieve selectivity for β-cleavage of APP and minimize adverse effects resulting from disrupted function of other BACE1 substrates. Importantly, impairment of eNOS/NO signaling, (common feature of endothelial dysfunction induced by cardiovascular risk factors), significantly increases expression of BACE1 and production of Aβ in human cerebrovascular endothelium.¹¹ In contrast, activation of soluble guanylate cyclase by NO exerts strong inhibitory effect on expression of BACE1.¹¹ Ongoing efforts to harness eNOS/NO signaling in prevention of cerebrovascular disease and cognitive impairment caused by AD resulted in discovery and clinical testing of central nervous system penetrant soluble guanylate cyclase stimulator, zagociguat.¹⁰⁷ However, Phase 2 clinical trial (NCT04798989) with zagociguat in patients with AD and associated vascular pathology was terminated because of problems with enrolment.¹⁰⁸ Nevertheless, based on the preclinical studies discussed in this review, it appears likely that stimulators of soluble guanylate cyclase may exert beneficial effects on cerebrovascular function in part by preventing pathological upregulation of BACE1.

7. Concluding remarks and future perspectives

Under pathological conditions upregulation of endothelial BACE1 and shift towards β-processing of APP exerts detrimental effects on eNOS/NO signaling and significantly impairs vasodilator and neurovascular protective function of NO. Recent identification of occludin as substrate for proteolytic activity of BACE1 in cerebrovascular endothelium, supports the concept that besides detrimental effect on eNOS signaling, excessive activity of endothelial BACE1 disrupts BBB. Thus, endothelial BACE1 appears to be viable therapeutic target in prevention and treatment of cerebrovascular pathology. This is consistent with existing evidence suggesting that preservation of endothelial function may help protect and preserve neuronal function and healthy cognition.

Several questions remain to be addressed in the future studies. First, advances in understanding of how and when cardiovascular risk factors may affect expression and function of BACE1 in endothelium will be important for further development and optimization of therapeutic interventions. Second, the exact molecular mechanisms responsible for control of BACE1 mRNA and protein expression in endothelium should be better defined. Third, further efforts should focus on discovery of previously unrecognized substrates for proteolytic activity of BACE1 in endothelium. Finally, further improvements in understanding mechanisms of BACE1-induced endothelial dysfunction may provide basis for development of new pharmacological approaches to preservation of healthy cerebrovascular function.

Highlights.

• Beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is expressed in endothelial cells of cerebral and systemic blood vessels.

• Several mechanisms are responsible for detrimental effects of excessive activation of endothelial BACE1 under pathological conditions: proteolytic cleavage of APP resulting in generation of cytotoxic concentrations of amyloid-β (Aβ) peptides, BACE1-induced inhibition of expression and function of glypican-1 and endothelial nitric oxide (eNOS), and proteolytic cleavage of occludin leading to disruption of blood-brain barrier function.

• Increased expression of BACE1 in endothelium is emerging as a novel mechanism of endothelial dysfunction induced by hypertension, diabetes, and hypercholesterolemia.

Nonstandard Abbreviations and Acronyms

AD

Alzheimer’s disease

Aβ

amyloid beta

APP

amyloid-precursor protein

AMPs

antimicrobial peptides

BACE1

β-site APP-cleaving enzyme

BBB

blood-brain-barrier

CD36

cluster of differentiation 36

CAA

cerebral amyloid angiopathy

cSVD

cerebral small vessel disease

Cov-2

coronavirus

COVID-19

corona virus disease of 2019

eNOS

endothelial nitric oxide synthase

GMP

guanosine monophosphate

GPC-1

glypican-1

JAM-A

junctional adhesion molecule A

NO

nitric oxide

OSA

obstructive sleep apnea

PGC-1α

PPAR-γ-coactivator 1-α

PPAR-γ

peroxisome proliferator-activated receptor-γ

SARS

severe acute respiratory syndrome

SIRT1

Sirtuin 1

TNF-α

tumor necrosis factor-α

ZO-1

zonula occludens-1