A wide range of single-cell transcriptomics studies of young versus aged disease-free mouse brains revealed endothelial aging-associated transcriptional profiles [
16,
26‐
31]. While some of these studies report that the overall abundance of ECs, or their arteriovenous zonation patterns, were not changed between young and aged animals [
16,
29,
31], others reported a decrease in capillary-venous ECs (i.e., EC subtype with mixed distribution of both capillary and venous marker genes) in brain tissue from old animals [
30]. In terms of aging-associated gene expression changes, a common finding in these studies was a typical increase in expression for the majority of differentially expressed genes (DEGs) upon aging, rather than a decrease [
16,
30]. Commonly upregulated genes in aged BECs include those related to inflammation and the immune response (e.g.,
Vcam1, B2m, Cxcl12, Oas1) and members of the interferon (IFN) signaling pathway (e.g.,
Stat1, Bst2, Ifnar1,
Gbp6 [
16,
29,
31,
32]), in line with chronic inflammation as a well-established hallmark of aging [
1]. These changes may be associated with age-dependent BBB dysfunction, as increased endothelial expression of pro-inflammatory cytokines has been linked with reduced expression of EC tight junction proteins [
33] and BBB permeability [
34]. Findings of decreased expression of classical BBB junction genes (e.g.,
Ocln, Cldn5) [
30] in aged BECs are in line with this. In some studies, aged BECs also commonly exhibited upregulation of genes related to the hypoxia response (e.g.,
Hif1a, Ldha, Aldoa), and genes involved in oxidative stress pathways (e.g.,
Sod1, Alpl,
Apoe), indicating an increased cellular stress response [
16,
28]. Increased oxidative stress in BECs has been associated with age-induced impairment of the cerebral microvascular system and neurovascular uncoupling [
13,
24,
35], age-related alterations of vascular reactivity [
36] and reduced pericyte coverage of cerebral microvessels [
37,
38], which could ultimately contribute to neuronal cell death [
15]. Another common finding was the age-related upregulation of
Vwf, a key regulator of hemostatic control, various Krüppel-like factors (e.g.,
Klf2, Klf4, Klf6), important for vasoprotection and response to injury, and genes involved in cell adhesion and/or extracellular matrix (ECM) organization (e.g.,
Adamts1, Itga6, Cyr61) [
16,
28,
31]. Angiogenic signaling pathways, such as insulin-like growth factor-1 receptor (IGF-1R) , vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-β),were also upregulated in aged BECs, indicated by the upregulation of genes such as
Igf1r,
Kdr,
Flt1,
Edn1,
Eng,
Id1,
Bmpr2, and
Acvrl1 [
16,
28,
30,
31]. These findings are in contrast with the well-known phenomenon of capillary rarefaction in aging tissues [
39], including the brain [
40], suggesting that these genes may be activated in a counteracting attempt to restore the lost vascular density. Besides enhancing angiogenesis, VEGF signaling has however also been associated with BBB disruption [
41,
42]. Aged BECs furthermore showed an upregulation of glycolytic genes (e.g.,
Pkm,
Aldoa,
Ldha)
, known to be important for angiogenesis, and which could indicate a shift toward glycolytic metabolism [
16]. Altered metabolism is a well-known characteristic of EC aging and senescence (Box
1). Caution is nevertheless warranted, as Zhao and colleagues reported a selectively decreased expression for several metabolic genes involved in glucose/energy metabolism and ATP synthesis in aged brain capillaries [
30]. Furthermore, Jin and colleagues reported decreased levels of
Aplnr in aged mouse brain ECs, potentially impairing angiogenesis in healthy aging and after stroke [
43]. BECs additionally exhibited an age-associated increase in protein synthesis, as demonstrated by an enrichment of genes involved in the proteostasis pathway [
28] and the upregulation of ribosomal subunit genes, such as
Rpl37, Rps20, and
Rps21 [
16,
31]. A slight age-associated increase of senescent BECs was furthermore reported in a scRNA-seq study of the mouse brain [
26]. The accumulation of senescent ECs has been directly connected to BBB dysfunction in vitro and in vivo [
44], at least in part via induction of the senescence-associated secretory phenotype [
15], and the dysregulation of nitric oxide signaling in BECs [
45].
Of note, age-related changes of the BEC transcriptomic profile were reversible by infusion with plasma derived from young mice [
16] or by heterochronic parabiosis (a surgical procedure where young and old mice are joined together to share a common circulatory system) [
28]. Here, capillary BECs were found to be the most susceptible cell type in the mouse brain to the aging or rejuvenating effects of old and young blood or plasma, respectively [
16,
28]. Moreover, exercise has been shown to exert beneficial effects on brain microvascular perfusion [
46,
47], and single-cell profiling revealed a putative endothelial-specific role in this phenomenon [
27]. Precisely, subventricular zone cell types in the mouse brain were shown to interact with ECs via TWEAK signaling and this interaction was lost in aged mice, likely driven by a loss of
Tnfrsf12a expression in ECs. Exercise was associated with the restoration of
Tnfrsf12a expression in ECs, suggesting this signaling axis to be at least one of the underlying drivers of the beneficial effect of exercise on health of the brain vascular endothelium [
27]. Lastly, glucagon-like peptide-1 receptor agonist treatment (known to alleviate various cellular phenotypes of aging) was also shown to improve BBB integrity and reverse, at least in part, aging-associated gene expression changes in brain ECs of aged mice [
30]. This again showcases the adaptable nature of aging-related gene expression changes in the aged brain endothelium.