Scientists Discover a Common Distress Signal in the Blood-Brain Barrier

Whether in the wake of a stroke, seizure, massive neuroinflammation, or a blow to the head, the endothelial cells of the blood-brain barrier respond with remarkable similarity, according to a study published in the November issue of Nature Neuroscience. Researchers led by Richard Daneman at the University of California, San Diego, reported that the endothelial cells that make up the BBB normally express a suite of genes that distinguishes them from the endothelia of other organs. However, BBB cells damaged in various ways lost this specialized signature, changing over to an expression profile more akin to endothelial cells in other parts of the body. The findings suggest that common mechanisms of BBB dysfunction underlie different brain injuries and diseases.

  • Endothelial cells that form the BBB have a distinct gene-expression profile.
  • It responded similarly to stroke, seizure, head trauma, neuroinflammation.
  • This “BBB dysfunction signature” mirrored endothelial gene expression in other organs.

David Attwell of University College London called the study exciting, noting that the similarities in gene-expression changes evoked by different insults was surprising. “This raises the possibility that successfully preventing (or increasing) endothelial cell gene-expression changes that occur in one disease may lead to a potential therapy for other types of CNS disorders,” he wrote.

“This exciting study provides a comprehensive data resource on the molecular underpinnings of brain endothelial cell dysfunction,” wrote Tony Wyss-Coray and Andrew Yang of Stanford University in a joint comment to Alzforum. “It was not known a priori whether such a core dysfunction module existed, and its identification opens a variety of fascinating questions.”

Tasked with shielding the precious brain from toxic insults while allowing crucial nutrients to cross, the blood-brain barrier is highly selective. Ergo, the endothelial cells that line the brain’s vessels are highly specialized, forming ultra-tight junctions and mobilizing molecular transporters not typically found in vessels supplying other organs. Disruption of the barrier is thought to play a hand in the pathogenesis of multiple injuries and diseases—including traumatic brain injury, stroke, seizures, and neurodegenerative disease such as Alzheimer’s (Mar 2008 news; Feb 2015 webinar; Jan 2019 news). 

What molecular shenanigans compromise the barrier? And are they similar across neurological diseases? Co-first authors Roeben Munji and Allison Soung and colleagues addressed these questions by comparing the transcriptomes of endothelial cells. Before diving into injury or disease models, the researchers compared the gene-expression profiles of healthy endothelial cells taken from the brain and other organs of mice. Compared with their counterparts from the kidney, lung, and liver, endothelial cells from the brain expressed a unique set of genes that encode metabolic enzymes and components of tight junctions, transporters, and the extracellular matrix, as well as units of the Wnt-β-catenin pathway known to support the BBB’s formation and maintenance. In contrast, endothelial cells in other organs of the body expressed a plethora of genes involved in immune functions, including receptors that latch onto leukocytes and whisk them across vessel walls.

How do brain endothelial cells change in the face of injury or disease? The researchers tracked gene-expression changes following four different insults known to disrupt the barrier: seizures, stroke, trauma, and experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. They induced seizures by injecting kainic acid, triggered stroke by occluding the middle cerebral artery, caused traumatic brain injury by dropping weights onto the heads of anesthetized mice, and set off EAE by injecting the 35-55 amino acid fragment of myelin oligodendrocyte glycoprotein, which elicited an inflammatory autoimmune response.

They then tracked the permeability of the barrier at three time points. In the earliest, or “acute” phase of each injury, the researchers found only minimal disruption of the BBB. However, in the so-called “subacute phase,” which was one or two days later depending on the model, the leakiness of the BBB reached its peak. About a month later, in the “chronic phase,” the barrier had partially or fully regained its integrity.

Each injury induced a bevy of gene-expression changes in brain endothelial cells. They varied substantially among injuries in the acute phase, but shared striking commonalities in the subacute phase, when the barrier was leakiest. By the chronic phase, gene expression had largely returned to normal in the stroke, seizure, and TBI models, but remained highly altered in the EAE model. Interestingly, the researchers found that the genetic signature of the healthy BBB endothelium was most downregulated in the acute phase of TBI, and the subacute phase of stroke, seizure, and EAE. Conversely, genes expressed predominantly in endothelial cells outside of the brain were turned up in the brain endothelium at these time points.

United in Dysfunction. A core group of 54 genes were upregulated in the subacute phase of all four disease models, while 136 (red numbers) were turned up in at least three. [Courtesy of Munji et al., Nature Neuroscience, 2019.]

At the subacute time point—when the barrier was leakiest across all models—54 genes were upregulated in common across all four models, and 136 were turned up in at least three models (see image at right). The researchers defined these 136 genes as a common BBB dysfunction module. It contained 68 genes normally expressed by endothelial cells outside of the brain, including many that belong to pathways involved in angiogenesis and inflammation.

Together, the findings suggest that while brain endothelial cells may initially respond differently to unique insults, they soon converge on a gene-expression profile that resembles those of endothelial cells in other organs of the body, said Daneman. Among other functions, these gene-expression changes likely ramp up interactions between the endothelium and circulating immune cells, he added.

The findings jibe with a recent study led by Wyss-Coray, which reported the increased expression of the leukocyte adhesion receptor VCAM1 on brain endothelial cells with age (Jul 2018 conference news; May 2019 news). Aging also weakens the BBB.

Important unanswered questions were whether the gene-expression changes Mungi and colleagues identified alter the integrity of the blood-brain barrier, and whether they are helpful or harmful to the brain, Daneman said. The full genetic dataset will be available to researchers at NCBI’s Gene Expression Omnibus. 

Will these findings extend to neurodegenerative diseases such as AD? Dementias typically have a much longer prodromal phase than the acute injuries modeled here, and the blood-brain barrier likely erodes more slowly. Some studies have been unable to detect overt breaches in the BBB in AD (Oct 2015 news). This important question is challenging to address, Daneman said, not only because the BBB is more intact in these diseases, but also because mouse models of AD poorly recapitulate the cerebrovascular pathologies associated with the disease. His lab is developing techniques to isolate endothelial cells from postmortem brain tissue of AD and PD patients. Daneman is also looking for serum biomarkers of BBB disruption.

Axel Montagne of the University of Southern California, Los Angeles, wrote that the paper could spur an investigation of how different cells in the brain’s vasculature, such as pericytes and smooth muscle cells, change when the BBB is compromised, and how gene-expression profiles of cells compare throughout the cerebrovascular tree, and in different regions of the brain (Vanlandewijck et al., 2018). Montagne envisions doing cerebrovascular-profiling studies in mouse models of AD.

“We also wonder whether this [BBB dysfunction] module will be found in chronic settings of neurodegeneration and aging,” wrote Yang and Wyss-Coray. “A hint arises in the module’s de-enrichment of BBB-specific genes, suggesting a common perturbation in surrounding mural-cell signals, such as from a loss of pericytes,” they added. “As pericyte loss has been reported in Alzheimer’s disease, this module may indeed be an even more generalizable hallmark of BBB dysfunction.”

Giuseppe Faraco of Weill Cornell Medical College in New York wondered whether early changes to brain endothelial function that precede overt BBB disruption might play a role in the pathogenesis of neurodegenerative diseases. Faraco recently reported that in response to a high-salt diet, dysfunctional brain endothelial cells trigger tau aggregation within the parenchyma that might wreak havoc on the brain (Oct 2019 news). Others have reported that BBB weakness itself correlated with dementia, regardless of ongoing tau or Aβ pathology (Jan 2019 news). 

The study underscores the emerging view that the BBB is a transcriptionally dynamic sensor of a variety of environmental stimuli, and that its gene products may functionally affect overall brain health, noted Yang and Wyss-Coray. “Whether these ‘sense-and-response’ capabilities decline with normal aging and neurodegenerative disease may be an important area of study,” they wrote.—Jessica Shugart

Comments

  1. This is an exciting paper from the Daneman group that studies how the unique characteristics of endothelial cells (ECs) around brain capillaries change in different types of pathology, in such a way that the ECs start to resemble ECs on peripheral capillaries.

    This is important because the blood-brain barrier (BBB) is normally maintained by special properties of CNS ECs: The presence of tight junctions between ECs and a suppression of transcytosis across the cells. BBB failure occurs in various types of brain pathology and contributes to neural dysfunction. In some circumstances, this failure may also provide a route for therapeutic agents to enter the CNS. Surprisingly, the paper shows that in different types of pathology there are similarities in how the gene-expression properties of the ECs alter (measured at the mRNA level: Future work will be needed to examine changes at the protein level, and also tease out the functional significance of the change in each of the many proteins whose expression is altered). This raises the possibility that successfully preventing (or increasing) EC-gene-expression changes that occur in one disease may lead to a potential therapy for other types of CNS disorder.

    View all comments by David Attwell

  2. This is a noteworthy study by the Daneman lab comparing the transcriptional profiles of mouse blood-brain barrier (BBB) endothelial cells (ECs) with those in other organs of the body (i.e., heart, lung, kidney, and liver) using RNA-Seq method and an inducible endothelial-specific reporter mouse model, ROSA-tdTomato;VE-Cadherin-CreERT2.

    First, they identified Wnt/β-catenin-related pathways (known to be crucial for BBB formation and maintenance), different transport mechanisms, and amino acid metabolism as key BBB-enriched pathways. The authors also pinpointed highly expressed tight-junction molecules including several that are BBB-enriched compared to the peripheral ECs such as Igsf5, Ocln, Lsr, Marveld2, Cgnl1, and Amot. In addition, they found BBB-enriched transporters including ATP-binding cassette (ABC) transporters (e.g., Abcb1a, encoding P-glycoprotein) and other transporters of energy metabolites (e.g., the famous glucose transporter GLUT1 encoded by the Slc2a1 gene), amino acids, neurotransmitters, ions, and others. I agree with the authors that BBB-enriched transcriptome data will provide a better understanding of BBB-specific functions as well as identifying targets to enhance drug delivery into the central nervous system.

    Importantly, the authors have also looked at disease models of stroke, traumatic brain injury, multiple sclerosis (MS), and seizures, each having profound BBB dysfunction. They noticed similar patterns of endothelial gene expression across all models in the acute/subacute phase, although each disease is caused by a unique trigger. Twelve common genes included Adamts4 (encoding ADAMTS4, a metalloproteinase), Atp8b1, Cd14, Ch25h, Kit, Lrg1, Pdlim1, Scgb3a1, Sele (encoding E-selectin, a leukocyte adhesion molecule), Tmem173, Trp53i11, and Upp1. These data suggest that finding a treatment limiting BBB dysfunction in one of the diseases may lead to treatment for others.

    There is no doubt that this exciting paper will lead to additional studies which should consider the following:

    1. The link between endothelial-gene profile and functional protein expression is missing here. Future studies should perform these experiments that are critical to better understand the BBB properties in health and disease.
    2. The newly discovered concept of “vascular zonation” or “BBB zonation” which refers to the heterogeneity of molecular and phenotypic changes of endothelial and mural—i.e., pericytes and vascular smooth muscle cells (VSMCs)—cells along the cerebrovascular tree (Vanlandewijck et al., 2018). In fact, there is increasing evidence for genetic and functional heterogeneity of these cells depending upon their location on the cerebrovascular bed, but also their geolocation in the brain (e.g., white vs. gray matter regions, hippocampus vs. cortical mantle, etc.).
    3. The transcriptional profiles of other vascular cell types, including pericytes, VSMCs, and astrocytes. Each vascular cell type can be FACS-sorted, and their respective transcriptional profiles investigated. One would think that they have distinct spatiotemporal patterns of gene expression in both health and disease. It would be interesting to dissect all these gene profiles in the proposed disease models and see whether a certain cell type has specific gene-expression changes prior to others.
    4. AD mouse models, since vascular dysfunction has become an important etiological factor (Montagne et al., 2017). Profiling the mouse brain vascular cell transcriptome in AD mice would be of interest, not only for the endothelial gene profile, but also for pericytes and VSMCs profiles as well. Indeed, damage to brain capillary pericytes has been shown to contribute to AD pathophysiology in mice (Montagne et al., 2017), and to develop early in older adults with cognitive impairment (van de Haar et al., 2016; Nation et al., 2019). 

    Finally, the authors have found that the Vcam1 gene (encoding for an inflammatory leukocyte adhesion molecule called VCAM-1) was mostly expressed in peripheral vessels, except in the experimental autoimmune encephalomyelitis mouse model of MS where Vcam1 was detected in brain vessels. Given that VCAM-1 is not constitutively expressed at the BBB, it would be interesting to investigate whether brain endothelium activation (e.g., increased Vcam1 at the BBB) occurs early in AD using Daneman’s BBB-enriched RNA-Seq method, and whether it precedes amyloid buildup, neurodegeneration, and memory deficits. Recent clinical studies revealed that soluble VCAM-1 was the top protein among 31 that increased with age (Yousef et al., 2019) and that higher levels of plasma sVCAM-1 are found in AD cases compared to age-matched controls (Huang et al., 2015). 

    References:

    van de Haar HJ, Burgmans S, Jansen JF, van Osch MJ, van Buchem MA, Muller M, Hofman PA, Verhey FR, Backes WH. Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease. Radiology. 2016 Nov;281(2):527-535. Epub 2016 May 31 PubMed.

    Huang CW, Tsai MH, Chen NC, Chen WH, Lu YT, Lui CC, Chang YT, Chang WN, Chang AY, Chang CC. Clinical significance of circulating vascular cell adhesion molecule-1 to white matter disintegrity in Alzheimer's dementia. Thromb Haemost. 2015 Nov 25;114(6):1230-40. Epub 2015 Aug 20 PubMed.

    Montagne A, Zhao Z, Zlokovic BV. Alzheimer's disease: A matter of blood-brain barrier dysfunction?. J Exp Med. 2017 Nov 6;214(11):3151-3169. Epub 2017 Oct 23 PubMed.

    Nation DA, Sweeney MD, Montagne A, Sagare AP, D'Orazio LM, Pachicano M, Sepehrband F, Nelson AR, Buennagel DP, Harrington MG, Benzinger TL, Fagan AM, Ringman JM, Schneider LS, Morris JC, Chui HC, Law M, Toga AW, Zlokovic BV. Blood-brain barrier breakdown is an early biomarker of human cognitive dysfunction. Nat Med. 2019 Feb;25(2):270-276. Epub 2019 Jan 14 PubMed.

    Vanlandewijck M, He L, Mäe MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Laviña B, Gouveia L, Sun Y, Raschperger E, Räsänen M, Zarb Y, Mochizuki N, Keller A, Lendahl U, Betsholtz C. A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018 Feb 14; PubMed.

    Yousef H, Czupalla CJ, Lee D, Chen MB, Burke AN, Zera KA, Zandstra J, Berber E, Lehallier B, Mathur V, Nair RV, Bonanno LN, Yang AC, Peterson T, Hadeiba H, Merkel T, Körbelin J, Schwaninger M, Buckwalter MS, Quake SR, Butcher EC, Wyss-Coray T. Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1. Nat Med. 2019 Jun;25(6):988-1000. Epub 2019 May 13 PubMed.

    View all comments by Axel Montagne

  3. This exciting study provides a comprehensive data resource on the molecular underpinnings of brain endothelial cell dysfunction. It builds on the group's prior work providing a healthy blood-brain barrier (BBB) transcriptome, an already incredibly useful resource. The authors describe here a core dysfunction module, a set of genes that become upregulated across four distinct and acute insults, with several implicated in human disease. It was not known a priori whether such a core dysfunction module existed, and its identification opens a variety of fascinating questions.

    For example, it will be important to determine which of, and how, the module genes are functionally linked to observed phenotypes, such as BBB leakiness. We also wonder whether this module will be found in chronic settings of neurodegeneration and aging. A hint arises in the module's de-enrichment of BBB-specific genes, suggesting a common perturbation in surrounding mural cell signals such as from a loss of pericytes. As pericyte loss has been reported in Alzheimer's disease, this module may indeed be an even more generalizable hallmark of BBB dysfunction. And with single-cell sequencing (scRNA-Seq) technologies increasingly adopted, it will be interesting to deduce vessel segment-specific changes across insults: It may be that their stroke model differentially impacts arterial cells compared to the rest of the endothelium, and so on. 

    It is becoming increasingly clear that the BBB is a transcriptionally dynamic sensor of a variety of environmental stimuli, and that its gene products may functionally affect overall brain health. Whether these “sense-and-response” capabilities decline with normal aging and neurodegenerative disease may be an important area of study.

    —Andrew Chris Yang is a co-author of this comment.

    View all comments by Tony Wyss-Coray

  4. This is an interesting study providing useful data for the understanding of brain endothelial function in both physiological and pathological conditions. The study shows that in different models of neurodegenerative diseases, the transcriptional responses of brain endothelial cells are similar. Although the acute gene expression response seems to be different in each model, the changes are similar in the subacute phase, suggesting that the response of brain endothelial cells to injury might be the same despite the nature of the insult. The data are intriguing and to a certain degree surprising since the pathophysiologies of these conditions are different.

    In addition, even if we consider that dysfunction of the blood-brain barrier (BBB) occurs in all the conditions that were studied in the paper, the time course and the mode of the BBB disruptions are different. Also, it is interesting to note that the response of brain endothelial cells to injury might involve acquiring a “peripheral” phenotype. Lastly, considering recent data showing gene-expression signatures in different sections of the cerebrovascular tree, in future studies it will be interesting to understand whether the transcriptional changes of brain endothelial cells in response to injury are common to arteries, capillaries and veins. Equally interesting will be to understand whether changes to brain endothelial function prior to disease onset and BBB disruption might play a role in the pathogenesis of neurodegenerative diseases. In this regard, it is worth mentioning that a dysfunction of endothelial cells is not necessarily associated with BBB dysfunction. For example, we have shown that a deficit in the ability of brain endothelial cells to produce nitric oxide induces cognitive dysfunction through tau aggregation, despite an intact BBB (Faraco et al., 2018; Faraco et al., 2019). 

    References:

    Faraco G, Brea D, Garcia-Bonilla L, Wang G, Racchumi G, Chang H, Buendia I, Santisteban MM, Segarra SG, Koizumi K, Sugiyama Y, Murphy M, Voss H, Anrather J, Iadecola C. Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response. Nat Neurosci. 2018 Feb;21(2):240-249. Epub 2018 Jan 15 PubMed.

    Faraco G, Hochrainer K, Segarra SG, Schaeffer S, Santisteban MM, Menon A, Jiang H, Holtzman DM, Anrather J, Iadecola C. Dietary salt promotes cognitive impairment through tau phosphorylation. Nature. 2019 Oct;574(7780):686-690. Epub 2019 Oct 23 PubMed.

    View all comments by Giuseppe Faraco

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References

News Citations

  1. Research Brief: SOD1 Mutants Cause Early Vascular Changes
  2. Absent Aβ, Blood-Brain Barrier Breakdown Predicts Cognitive Impairment
  3. VCAM1: Gateway to the Aging Brain?
  4. Paper Alert: VCAM1 Opens the Door to Brain Aging
  5. Does the Blood-Brain Barrier Stand Up to Alzheimer’s? Study Finds No Breach
  6. Not Just Blood Pressure—Dietary Salt Linked to Tau Phosphorylation

Webinar Citations

  1. Leaky Blood-Brain Barrier a Harbinger of Alzheimer's?

Paper Citations

  1. Vanlandewijck M, He L, Mäe MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Laviña B, Gouveia L, Sun Y, Raschperger E, Räsänen M, Zarb Y, Mochizuki N, Keller A, Lendahl U, Betsholtz C. A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018 Feb 14; PubMed.

External Citations

  1. NCBI’s Gene Expression Omnibus

Further Reading

No Available Further Reading

Primary Papers

  1. Munji RN, Soung AL, Weiner GA, Sohet F, Semple BD, Trivedi A, Gimlin K, Kotoda M, Korai M, Aydin S, Batugal A, Cabangcala AC, Schupp PG, Oldham MC, Hashimoto T, Noble-Haeusslein LJ, Daneman R. Profiling the mouse brain endothelial transcriptome in health and disease models reveals a core blood-brain barrier dysfunction module. Nat Neurosci. 2019 Oct 14; PubMed.

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