The Role of PRRC2B in Cerebral Vascular Remodeling Under Acute Hypoxia in Mice

Abstract High altitude exposure leads to various cognitive impairments. The cerebral vasculature system plays an integral role in hypoxia‐induced cognitive defects by reducing oxygen and nutrition supply to the brain. RNA N6‐methyladenosine (m6A) is susceptible to modification and regulates gene expression in response to environmental changes, including hypoxia. However, the biological significance of m6A in endothelial cell performance under hypoxic conditions is unknown. Using m6A‐seq, RNA immunoprcipitation‐seq, and transcriptomic co‐analysis, the molecular mechanism of vascular system remodeling under acute hypoxia is investigated. A novel m6A reader protein, proline‐rich coiled‐coil 2B (PRRC2B), exists in endothelial cells. PRRC2B knockdown promoted hypoxia‐induced endothelial cell migration by regulating alternative splicing of the alpha 1 chain of collagen type XII in an m6A‐dependent manner and the decay of matrix metallopeptidase domain 14 and ADAM metallopeptidase domain 19 mRNA in an m6A‐independent manner. In addition, conditional knockout of PRRC2B in endothelial cells promotes hypoxia‐induced vascular remodeling and cerebral blood flow redistribution, thus alleviating hypoxia‐induced cognitive decline. Therefore, PRRC2B is integral in the hypoxia‐induced vascular remodeling process as a novel RNA‐binding protein. These findings provide a new potential therapeutic target for hypoxia‐induced cognitive decline.


Figure S1 .
Figure S1.Related to Figure 1.PRRC2B is a novel m6A reader.A. The immunoprecipitation efficiency of PRR2CB-HA by HA antibody.B. The correlation of 3 independent repeats of m6A-seq.C. Pie chart depicting the distribution of m6A modification peaks.D. Distribution of m6A modification peaks across the length of mRNA.E. Classic m6A modification motif identified by HOMER, p=1e-6.F. PRRC2B binding gene containing m6A modification.

Figure S2 .
Figure S2.Related to Figure 3. PRRC2B regulates target gene fate decision.A. Schematic illustration of PRRC2B binding protein screening.B. The IP efficiency was detected by western blot.C. Sliver staining of the total proteins after immunoprecipitation.D-E.GO analysis (D) and KEEG analysis (E) of PRRC2B binding proteins enrichment pathway.F. The knockdown efficiency of si-METTL3 in HUVECs.G.The different transcriptional variants expression level in METTL3 knockdown HUVECs.H-I.The pre-mRNA and mature mRNA of MMP14 (F) and

Figure S3 .
Figure S3.Related to Figure 4. PRRC2B downregulated in acute hypoxia exposure.A. The mRNA levels of PRRC2B in HUVEC across hypoxic times were detected by real-time qPCR.(Student's t-test, ***p < 0.001).B. Western blot detected the protein levels of PRRC2B in HUVEC across hypoxic times.C. Schematic of adult cerebral endothelial cells separation procedure.D. The expression level of marker genes of endothelial cells in separated cell categories.E. Flow cytometry assay of sorted cells.F-G.The mRNA level and (F) the protein level (G) of PRRC2B in mice cerebral endothelial cells upon hypoxia stimulation.H. COL12A1 transcriptional variant in hypoxia in HUVECs.I.The gene expression level in cerebral endothelial cells in Ctrl and cKO mice in hypoxia and normoxia.One-way ANOVA followed the Tukey test, ***p < 0.001, N=3.

Figure S4 .
Figure S4.Hypoxia-induced cerebral vascular remodeling. A. Vascular system in the hippocampus was imaged by brain clarity methods, and blood vessels were labeled by TD-tomato fluorescence.B. The Imaris workflow of vascular data analysis captured by light sheet microscopy.C. Statistic data of blood vessel diameter in each group.D. The total vessel length per volume.E. The volume fraction percentage in normoxia and hypoxia treatment.(Student's t-test, ***p < 0.001).

Figure S5 .
Figure S5.Related to Figure 5. Prrc2b conditional knockout aggregates hypoxia-induced vascular remodeling. A. The knockdown efficiency of Prrc2b in endothelial cells was determined by qPCR.B-F.Statistic data of vessel area percentage (B), vessel mean diameter (C), the total length of vessels (D), vessel junction numbers (E), and vessel volume (F) in each group of mice hippocampus.G. Representative image of cerebral blood vessels in the cortex.H. IB4 staining shows vasculature changes in the cortex, which is consistent with the results of TD-tomato-labeled mice.I-J.Statistic data of vessel area percentage (I) and vessel mean diameter (J) in IB4 staining image.(one-way ANOVA followed Tukey test, *p < 0.05, ***p < 0.001).