Investigation of Apelin signaling as  a regulator for cerebrovascular  specialization

Lukas Herdt

Thesis

Open Access

Investigation of Apelin signaling as a regulator for cerebrovascular specialization

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Date

2025-11-18

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Lukas Herdt

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Helker, Christian

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Borchers, Annette

Abstract

Organ-specific angiogenic signaling pathways establish a vascular network that meets the unique metabolic and functional demands of each tissue. The brain has the highest metabolic need of all organs accounting for approximately 20% of daily energy consumption, despite representing only 2% of total body weight. To adequately supply the brain with nutrients and oxygen, the central nervous system (CNS) must be highly vascularized. The vasculature in the brain is mainly characterized by the establishment of the blood-brain-barrier (BBB) that protects neural tissue by restricting the passage of harmful molecules and regulating nutrient exchange by the endothelial cells (ECs). To maintain CNS homeostasis and function, certain regions require specialized vascular permeability that is ensured by fenestrated blood vessels. These specialized ECs form pores, so-called fenestrae, within their cell membrane that regulate the molecular exchange across the endothelium. While signaling pathways required for BBB angiogenesis have been extensively studied over the past decades, we still lack knowledge about the molecular signals specifically required for fenestrated blood vessel formation. The Apelin receptor (Aplnr), a class A (rhodopsin-like) G protein-coupled receptor (GPCR) and its two endogenous peptide ligands, Apelin and Apela, has been shown to play key roles during the development of the cardiovascular system in vertebrates, including mice, frog and zebrafish. Recently, neurovascular Apelin signaling has been shown to promote vascularization of the spinal cord, which suggests a broader role of Apelin signaling in CNS vascularization. However, it remains unknow whether Apelin signaling is required for cerebral angiogenesis and cerebrovascular specialization. This work addresses the role of Apelin signaling during CNS vascularization with particular focus on cerebral angiogenesis and cerebrovascular specialization. For this purpose, we used the zebrafish as a model organism to decipher the cellular mechanisms underlying CNS vascularization mediated through the Apelin signaling pathway. Additionally, we engineered genetically encoded APLNR activity biosensors, that enable real-time monitoring of APLNR activity in model cell lines and in living organisms. By applying these biosensors in zebrafish larvae, we visualized the spatiotemporal bioavailability of Apelin across the brain. By generating novel transgenic reporter lines and analyzing publicly available single cell RNA sequencing datasets, we found aplnrb expression closely mirroring that of the canonical fenestration marker plvapb, suggesting Aplnrb as a novel marker for fenestrated ECs. By investigating apln and aplnrb mutant larvae, we could show that the Apelin signaling pathway is a crucial regulator of fenestrated capillary formation in the Choroid plexus (CP), while it is dispensable for adjacent BBB-forming capillaries and BBB establishment. Our genetic analysis and RNA sequencing results revealed a previously unrecognized meningeal-vascular signaling axis required for fenestrated blood vessel development. Undifferentiated, yet pre-programmed, leptomeningeal fibroblast progenitor cells are the primary source of the Apelin ligand, thereby regulating and guiding fenestrated capillaries in the CP. Furthermore, we generated genetically encoded APLNR conformational biosensors based on Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and the conformation-sensitive circularly permuted green fluorescent protein (cpGFP) to measure APLNR activity in real-time. After comparing the performance of various APLNR biosensor variants, we focused on two APLNR-cpGFP biosensor constructs due to their favorable signal-to-noise ratio. These variants were pharmacologically characterized in model cell lines and investigated for their in vivo application. Analysis of the biosensor functionality in living zebrafish embryos demonstrated that these biosensors can monitor endogenous Aplnr activity, respond specifically to the endogenous Aplnr ligands, and detect Apelin ligand gradient concentrations across cellular distances within a tissue. Moreover, by expressing the biosensor by meningeal fibroblasts, we observed Apelin ligand hotspots across the brain, that spatiotemporally coincided with fenestrated vessel sprouting as previously observed. In summary, we identified a meningeal-vascular signaling axis mediated by Apelin signaling that is specifically required for fenestrated capillary formation in the CP, but dispensable for adjacent BBB vessels. By using our newly generated APLNR activity biosensors, we demonstrated the spatiotemporal bioavailability of the Apelin ligand across the brain which is required for CP vascularization.