# Pericyte angiopoietin2 and neonatal intracranial hemorrhage

> **NIH NIH R21** · YALE UNIVERSITY · 2021 · $460,625

## Abstract

Project Summary / Abstract
 Intraventricular hemorrhage (IVH) is a debilitating condition at any age but is especially common and
devastating in premature infants as it is associated with substantial long-term morbidity (e.g., cerebral palsy,
cognitive deficits) and mortality in this population. In premature infants, most IVH stems from hemorrhage in
the germinal matrix (GM), a collection of highly vascularized neuronal and glial precursor located underneath
the ventricular ependyma. Pericytes (PCs) and endothelial cells (ECs) are key components of the blood-brain
barrier (BBB). In the GM, reduction in the numbers of PCs and levels of the ligand transforming growth factor
(TGF)-β1 in comparison to the white matter and cortex has been implicated in the high propensity of the GM to
hemorrhage. Indeed, we previously reported that deletion of the gene encoding activin receptor-like kinase 5
(ALK5), a type I TGFβ receptor, in PCs leads to gross GM hemorrhage (GMH)-IVH in embryonic mice (Dev
Cell, 44:665) largely through effects on ECs. However, mechanisms underlying these non-cell autonomous
effects on ECs are incompletely defined. To search for PC-derived factors that may signal to ECs, we
conducted bulk RNA-sequencing of human brain PCs with and without both siRNA Alk5 knockdown and
TGFβ1 treatment and identified the secreted factor angiopoietin (Angpt) 2. ANGPT2, which is broadly
implicated in EC tube destabilization and/or remodeling, is predominantly expressed by ECs, but PC
expression has not been reported. Moreover, the role ANGPT2, and certainly PC-derived ANGPT2, in GMH-
IVH has not been studied. Follow-up studies confirmed that TGFβ1 treatment of human brain PCs rapidly and
robustly down-regulates levels of Angpt2 mRNA (~65% and ~90% reduction in 3 h and 12 h, respectively) and
protein in an ALK5-dependent manner. This rapid reduction in Angpt2 mRNA suggests agonist-induced
transcript destabilization, which we will investigate in the proposed experiments. Furthermore, chromatin
immunoprecipitation assays revealed that TGFβ1 treatment of PCs promotes epigenetic and transcriptional
repression of Angpt2, increasing histone deacetylase-2 binding and reducing histone acetylation (H3K9ac,
H3K27ac) and RNA polymerase II binding to the proximal promoter. Finally, in mice with Alk5 deletion in PCs,
perivascular ANGPT2 expression is markedly increased. Thus, we hypothesize that when TGFβ-ALK5
signaling in PCs is disrupted, ANGPT2 levels are increased, destabilizing ECs and leading to disruption of the
BBB and GMH-IVH. We will use cultured human brain vascular cells, transgenic mice, proteomic and
3’UTR/microRNA screens and de-identified human brain samples to test this hypothesis in two specific aims:
1) determine mechanisms of TGFβ-mediated regulation of Angpt2 in PCs and effects on ECs; and 2) elucidate
role of PC ANGPT2 in GMH-IVH of mice and humans. This R21 will yield key insights into BBB formation and
GMH-IVH pathogenesis and form the foundatio...

## Key facts

- **NIH application ID:** 10288547
- **Project number:** 1R21NS123469-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Daniel Greif
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $460,625
- **Award type:** 1
- **Project period:** 2021-07-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10288547

## Citation

> US National Institutes of Health, RePORTER application 10288547, Pericyte angiopoietin2 and neonatal intracranial hemorrhage (1R21NS123469-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10288547. Licensed CC0.

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