Cell: The combination of endothelial and parietal cells promotes prenatal human cerebral angiogenesis

There is a growing recognition that the vascular system affects brain function
in health and disease.
Endothelial cells and parietal cells constitute the main structural and functional components of
blood vessels.
Endothelial cells form the lumen of blood vessels and also secrete factors
that affect neurogenesis in the mouse brain.
Parietal cells include smooth muscle cells and pericytes, located on the outside
of endothelial cells.
They are essential
for the formation of the blood-brain barrier (BBB) and neurovascular coupling.

Brain endothelial cells are thought to originate from the perineural vascular plexus outside the brain parenchyma, while the ontogenesis of parietal cells remains to be further clarified
.
Although recent studies have elucidated the contribution of vascular cells to brain aging and neurodegeneration, we lack a comprehensive understanding
of the stages of prenatal cerebral vascular cell development.

On September 29, 2022, Eric J.
Huang's research group at the University of California, San Francisco published a research paper entitled "Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain" in the journal Cell.
The study revealed the transcriptomic and functional properties of different endothelial and parietal cells in the primary blood vessels of the human brain, as well as the trajectory and heterogeneity
of blood vessels in human brain development.

Research results

1.
Angiogenesis in the prenatal human brain

To study angiogenesis in the prenatal human brain, the authors labeled endothelial cells with aCD31 antibody and NG2 and PDGFR-b antibodies to identify parietal cells in the cerebral cortex, including the cerebral cortex, ventricular area/subventricular region (VZ/SVZ) region, and subcortex, including ganglion protrusions (GE) at 14~39 weeks of gestation (GW) (Fig.
1A, 1B).

Given the presence of active neurogenesis in the cerebral cortex inVZ/SVZ and subcortical GE, the authors divided these regions into three regions
based on their distance from the ventricles.
Region 1 is defined by Nestin and PDGFR-β expression, including VZ and SVZ up to 100 μm in size, rich in radial glial cells
.
The second region is about 100~1μm away from VZ/SVZ, and immature neurons are densely packed
.
Finally, region 3 defines a 500 μm region at the far end of region 2 (Figure 1C).

The results show that at 14–17 GW, region 1 does contain more branch points than region 2-3 (Figure 1H).

In addition, at 14–17 GW, region 1 contains more Ki-67+ endothelial cells than region 3 (Ki-67+; CD31+) and parietal cells (Ki-67+; PDGFR-β+) (Figure 1I-1K).

These findings suggest that the neurogenic ventricles of the prenatal human brain are also highly angiogenic
.

Figure 1: Angiogenesis in the periventricular region of the stromal stromal brain in the prenatal human brain

2.
Analysis of cerebrovascular cells in the second trimester of pregnancy

To investigate the stage characteristics of vascular development in the second trimester, the authors employed a flow cytometry-based strategy to purify endothelial and parietal cells
.
Flow cytometry confirmed the isolation of these markers from different endothelial cell populations (CD45; CD31 +; and ANPEP) and parietal cell populations (CD45; CD31; and ANPEP+) (Figure 2A).

The results showed that, in total, we collected data from 147,228 cells from 7 samples (2 per region), reading an average of 96,778 reads per cell, sequencing depth of 1468 genes, and 122,798 unique molecules
per cell.
The visualization of scRNA-seq data from these samples by UMAP revealed significant separation
of endothelial and parietal cells.
Endothelial cells express PECAM1, CD34, and TIE1, and parietal cells ANPEP, PDGFRB, and RGS5, as expected (Figures 2E and 2F).

Figure 2: Single-cell transcriptomics results of fetal brain endothelial and parietal cells in the second trimester

3.
Endothelial cell development trajectories

UNBIASED CLUSTERING IDENTIFIED FIVE SUBTYPES OF 15–23 GW OF PRENATAL HUMAN CEREBRAL ENDOTHELIAL CELLS, INCLUDING MITOSIS, VENOUS DIVISION, CAPILLARY DIVISION, TIP CELL DIVISION, AND ARTERIAL DIVISION (FIGURES 3A, 3B).

Next, the authors used RNA velocity to analyze scRNA-seq data from endothelial cells to interpret the relationship between
different cell clusters and their developmental trajectories.
THE RESULTS SHOW THAT AT 15 GW, MITOTIC ENDOTHELIAL CELLS GIVE RISE TO VENOUS ENDOTHELIAL CELLS, WHICH THEN PROGRESS TO AN INTERMEDIATE STAGE CONSISTING OF CAPILLARY ENDOTHELIAL CELLS AND TIP CELLS, AND FINALLY TERMINATE AS ARTERIAL ENDOTHELIAL CELLS (FIGURE 3D).

Figure 3: Development of nascent vascular endothelial cells in the human brain

4.
Prenatal parietal cell development of the human brain

The authors performed unbiased clustering of the scRNA-seq dataset to identify four subtypes of parietal cells, including smooth muscle cells, classical pericytes, fibroblasts, and mitotic parietal cells (Figures 4A, 4B).

All four subtypes were detected in GE and cortex at 15-23 GW (Figure 4A).

Mitotic parietal cells express cell cycle-related genes, including AURKB, NUF2, BIRC5, KIF20B, UBE2C, and HHMR, which define GO terms such as cell cycle, cell cycle process regulation, and AuroraB pathway (Figures 4B, 4C).

。 Next, the authors analyzed the maturation trajectories of parietal cells using RNA velocities, showing three different cell maturation states of parietal cells at 15 GW, starting with mitotic parietal cells (brown, UBE2C+, and BIRC5+), progressing to non-proliferative smooth muscle cells (red, MYL9+, and TAGLN+), and ending with classical pericytes (blue, ATP1A2+, and KCNJ8+) (Figure 4D).

Figure 4: Development of parietal cells in the primary vascular system in the middle phase of the human brain

Taken together, the results of this study support a model in which the development of the vascular system in the second trimester consists of
a range of different endothelial and parietal cell subtypes.
The findings also provide evidence
that endothelial cell trajectories are guided by intrinsic programs and can be influenced by specific subtypes of environmental signals.
The scRNA-seq dataset revealed another interesting result, namely that in the early stages of the second trimester, endothelial cells showed more transcripts associated with oxidative phosphorylation, supporting the possibility that endothelial cells during this period could upregulate gene expression associated with oxidative phosphorylation to compensate for smaller mitochondrial masses
.