Macrophages Blamed for Vascular Trouble in ApoE4 Carriers

Series - International Conference on Alzheimer's and Parkinson's Diseases 2023:

Here’s a radical idea. Perivascular macrophages—those innate immune cells sitting oh-so-innocently on the small blood vessels in our brains—might be why ApoE4 carriers are more susceptible to cerebrovascular disease. So said Costantino Iadecola, Weill Cornell Medicine, New York, in his presentation at AD/PD 2023, held March 28 to April 1 in Gothenburg, Sweden. Iadecola reported that in mice, these macrophages both produce ApoE4 and react to it, all the while unleashing reactive oxygen species, aka free radicals, that curb cerebral blood flow. Iadecola believes that, in people who carry an ApoE4 gene, these cells might exacerbate cerebral amyloid angiopathy and ARIA, an inflammatory condition caused by Alzheimer's disease and anti-Aβ immunotherapy.

  • ApoE4 carriers face higher odds of cerebrovascular disease.
  • In mice, ApoE4, but not E3, restricts cerebral blood flow.
  • Perivascular macrophages release ApoE4, and respond to it.
  • They spew damaging reactive oxygen species.

Previously, Iadecola's group had reported that perivascular macrophages pump out reactive oxygen species in response to Aβ, putting the squeeze on endothelial cells and narrowing blood vessels (May 2017 news). What's more, they found that cerebral blood flow, endothelial function, and neurovascular coupling were restricted in ApoE4 targeted replacement mice (Koizumi et al., 2018).

Could perivascular macrophages explain these deficits, as well, even without any toxic Aβ present? “These cells are very much plastered on the surface of the arterioles of the brain, and they are loaded with ApoE receptors and with free radical-producing enzymes, so they are the perfect place to make radicals that then affect the blood vessels,” Iadecola told the audience.

To explore this idea, Antoine Anfray, Laibaik Park, and colleagues in the lab first studied ApoE in wild-type mice. They applied some of it directly to the brain surface through a hole in the skull, and then, through a glass window, observed how blood vessels in the whisker-barrel cortex responded. This part of the cortex is an established system for studying cortical responses to sensory stimulation. Blood flow and electrical field potentials increase there when individual whiskers of the mice are tickled.

Indeed, just as in the targeted replacement mice, ApoE4 reduced endothelial function and neurovascular coupling when applied to wild-type mouse cortex. ApoE3 did not. Lipidated ApoE4, a more potent form of this lipid-packing protein, had a slightly stronger effect, while RAP, an inhibitor of ApoE receptors, completely suppressed it.

Militant Macrophage. In the brain of a living mouse, a perivascular macrophage sitting on a blood vessel wall takes up blue dextran (left). Adding ApoE4 to the brain boosts the cell's production of reactive oxygen species, which are detected with the red fluorescent reporter dihydroethidine (DHE). ApoE3 has no effect (right). [Courtesy of Costantino Iadecola.]

Was it the perivascular macrophages? A series of in vivo and in vitro experiments suggested as much. Two-photon microscopy revealed PVMs as a major source of free radicals in response to ApoE4, while the NADPH oxidase inhibitor gp91ds prevented the vascular dysfunction. PVMs are loaded with NADPH oxidase, which is a major source of reactive oxygen species (ROS) in these cells.

Further hints of PVM involvement came from examining cells isolated from the brain. PVMs from ApoE4 targeted replacement mice produced more ROS than PVMs from wild-type. Anfray and Park saw no such spike in other cell types, including microglia, another major source of free radicals. More direct evidence came when the scientists ablated PVMs by injecting clodronate into the brain ventricles. This bisphosphonate drug was developed for osteoporosis. It tempers osteoclasts; however, when macrophages take it up, it induces apoptosis. In ApoE4 targeted replacement mice, clodronate slashed macrophages on the ipsilateral side of the brain to a 20th of their normal number; this completely rescued endothelial function and neurovascular coupling in response to whisker stimulation.

If PVMs are reacting to ApoE4, then where does it come from? Iadecola suspects the macrophages themselves. Single-cell transcriptomic analysis of myeloid cells from the mouse brain suggested that PVMs make six times more ApoE than do microglia, endothelial cells, or blood vessel mural cells. Still, that does not prove the cells are responding to “home-made” ApoE in a cell-autonomous fashion. To test this directly, scientists in his lab developed conditional knockouts by crossing macrophage-driven Cre recombinase mice with “floxed” ApoE3 or ApoE4 mice. Cre, which can be induced by adding tamoxifen in this system, removes genes flanked by flox sequences.

The crosses behaved exactly like TR mice until the recombinase was turned on. In other words, neurovascular coupling was suppressed in the ApoE4 mice, but not once the PVM ApoE got spliced out. Then the mice behaved like wild-type, indicating that the PVMs indeed were the source of the ApoE that had been causing their vascular trouble.

The icing on the cake? When the scientists added back ApoE4 to the brains of these knockouts, blood flow was once again suppressed, as was neurovascular coupling. “Everything else required for this dysfunction was present, except there was no ApoE4 to activate the necessary receptors,” said Iadecola.

As in Alzheimer's disease, there was an allele difference. Replacing ApoE3 PVMs with ApoE4 ones caused vascular dysfunction, while replacing ApoE4 PVMs with ApoE3 ones ameliorated it. This was done by irradiating mice to kill off endogenous macrophages and then giving them fresh bone marrow of the alternative genotype.

Other scientists at the meeting called the presentation impressive. Christian Haass, Ludwig Maximilians University in Munich, wondered if the PVMs upregulate ApoE4 in response to some sort of challenge, such as presence of Aβ in cerebral amyloid angiopathy, just as disease-associated microglia do in the Alzheimer’s disease brain parenchyma. “Absolutely, this would be our assumption,” said Iadecola. “Now that we have the knockout mice, we can cross them with other mice to test these hypotheses."—Tom Fagan

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References

News Citations

  1. Do Perivascular Macrophages Mediate Aβ Pathology?

Paper Citations

  1. Koizumi K, Hattori Y, Ahn SJ, Buendia I, Ciacciarelli A, Uekawa K, Wang G, Hiller A, Zhao L, Voss HU, Paul SM, Schaffer C, Park L, Iadecola C. Apoε4 disrupts neurovascular regulation and undermines white matter integrity and cognitive function. Nat Commun. 2018 Sep 19;9(1):3816. PubMed.

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