Aorta Cells e AngII: Origins Matter Less Than You’d Think Early On

Okay, so let’s talk about something pretty serious: thoracic aortopathy. That’s a fancy way of saying diseases affecting the upper part of your aorta, the big artery that pumps blood from your heart. Think aneurysms (bulges), dissections (tears), or even rupture – definitely not things you want happening! It’s a major health issue, and honestly, we don’t have great drugs to stop it from starting or getting worse. So, understanding *how* it happens is super important.

Turns out, the ascending aorta, the bit right near your heart, is particularly vulnerable. And the cells that make up its wall, mainly smooth muscle cells (SMCs) and fibroblasts, are key players. Here’s where it gets interesting: these cells in this specific region come from different places during embryonic development. Some are from the second heart field (SHF), and others are from the cardiac neural crest (CNC). We already had a hunch that SHF-derived cells were really important in the *later* stages of this disease, especially when a troublemaker molecule called Angiotensin II (AngII) is involved. But what about the CNC-derived cells? And what happens right at the very beginning, before the damage is obvious? That was a bit of a puzzle.

The Big Question: Do Origins Matter Early On?

My colleagues and I wanted to figure out if these cells, depending on where they came from embryonically (SHF or non-SHF, which in the ascending aorta is mostly CNC), respond differently to AngII right at the start of the problem. We also knew that trying to genetically mess with CNC cells (like deleting certain genes) often caused serious issues or even death in developing mice, making it tough to study their role in adult aortopathy. So, we needed a different approach to compare their early responses.

The Experiment: A Quick Look Inside Cells

So, what did we do? We used some clever mice where we could tag the SHF-derived cells with a green fluorescent protein (mGFP) and the non-SHF cells with a red one (mTomato). This is like giving them little colored badges based on their origin. We then gave these mice AngII for just three days. Why only three days? Because we wanted to catch the very early, “pre-pathological” changes – the stuff happening *before* the aorta starts looking sick.

After those three days, we harvested the ascending aortas. We carefully sorted the cells based on their colored badges using a technique called FACS (Fluorescence-Activated Cell Sorting). It’s like a super-fast, high-tech cell sorter! Then, we performed single-cell RNA sequencing (scRNAseq). This is a powerful tool that lets us read the “instruction manual” (the RNA transcripts) inside thousands of individual cells. By comparing the RNA profiles, we could see which genes were turning on or off in response to AngII in SHF cells versus non-SHF cells.

What the Data Showed: Smooth Muscle Cells

First up, we looked at the smooth muscle cells. These are the main cells in the middle layer of the aorta wall, responsible for its structure and flexibility. When we infused AngII, we saw significant changes in gene expression in *both* the SHF-derived and the non-SHF-derived SMCs. Lots of genes were affected!

But here’s the kicker: when we compared the changes *between* the two origins, the differences were actually pretty modest. Out of over 3,300 genes that changed, almost all of them changed in the same direction (up or down) in both SHF and non-SHF cells. And for most genes, the *amount* of change was very similar between the two groups.

We looked at genes involved in things like:

• Protein folding
• Cell adhesion
• Extracellular matrix (the stuff between cells that gives tissue structure)

Even some genes we thought might be different based on previous studies, like Dcn or Des, showed only small differences between origins in this early phase. Genes involved in breaking down the matrix (like Mmp2 and Mmp9) and even the important TGF-β signaling pathway didn’t show statistically significant differences between the origins in our interaction analysis. SMC contractile genes and major elastic fiber components like Eln (elastin) and Fbn1 (fibrillin 1) also had only modest differences between origins.

So, for the smooth muscle cells, at least in the very early stages of AngII exposure, their embryonic origin didn’t seem to dictate drastically different transcriptional responses.

What the Data Showed: Fibroblasts

Next, we turned our attention to fibroblasts. These cells are also crucial for maintaining the structure and integrity of the aortic wall, especially by producing and organizing the extracellular matrix.

Similar to the SMCs, short-term AngII infusion caused notable changes in gene expression in fibroblasts from *both* lineages (SHF and non-SHF). Over 2,900 genes were affected. And again, the vast majority (nearly 99%) changed in the same direction in both groups.

When we looked for differences *between* the origins, the overall picture was much like the SMCs: modest differences. We identified about 600 genes with potential interaction effects between AngII and origin, and these were primarily related to the extracellular matrix. But when we checked the magnitude of these differences, only a small percentage of genes showed a substantial change between the origins. Molecules previously linked to aortopathy, like Il33, Col3a1, and S100A4, also showed limited lineage-specific differences overall. Key players in TGF-β signaling and elastic fibers were either not significantly different or showed only minor variations between the origins.

The Plot Twist: A Special Fibroblast Group

Now, here’s where things got a little more interesting. In our previous work, we had spotted a specific sub-cluster of fibroblasts that seemed potentially harmful in AngII-driven aortopathy. We checked if this sub-cluster existed in both SHF and non-SHF lineages after AngII infusion, and indeed it did! These cells had a unique gene signature, including high levels of Pi16 and specific markers like Ube2c and H2afz.

Because these cells were quite rare at baseline, we focused on comparing them *between* the SHF and non-SHF origins specifically after the 3-day AngII infusion. And *in this specific group*, turns out there *were* some notable differences between the origins, particularly in genes related to the extracellular matrix.

For example:

• Some collagen genes (Col15a1, Col18a1) were *more* abundant in SHF-derived cells in this sub-cluster.
• But crucially, genes like Eln (elastin) and Fn1 (fibronectin 1), which are vital for elastic fibers, and other collagen genes (Col3a1, Col8a1), important for collagen fiber formation, were *less* abundant in the SHF-derived fibroblasts in this special sub-cluster compared to their non-SHF counterparts.

This was a bit of an “aha!” moment. While the overall fibroblast population showed only modest differences between origins, this specific, potentially problematic sub-cluster *did* exhibit lineage-specific alterations in key matrix genes.

Genetic defects in Eln and Col3a1 are linked to serious aortic problems in humans, so seeing reduced levels of their mRNA in SHF-derived fibroblasts within this specific sub-cluster is intriguing. It suggests these particular SHF-derived fibroblasts might be less capable of maintaining the structural integrity of the aorta’s matrix when hit by AngII, potentially contributing to weakness.

Putting It All Together e Looking Ahead

So, what’s the takeaway from all this? My analysis suggests that during the very early, pre-pathological phase of AngII-induced thoracic aortopathy, the overall transcriptional response of smooth muscle cells and fibroblasts in the ascending aorta is pretty similar regardless of whether they came from the SHF or other origins (mostly CNC).

However, there’s that interesting exception: a specific sub-cluster of fibroblasts, found in both origins, where the SHF-derived ones show reduced expression of critical extracellular matrix genes like Eln and Col3a1 compared to their non-SHF cousins.

This finding helps clarify things a bit. Previous studies, including our own, pointed to SHF-derived cells being major players in AngII-induced aortopathy. But those studies often looked at later stages of the disease. Our current work, focusing on the *initiation* phase, suggests that perhaps the early response to AngII is broadly similar across origins, but the SHF-derived cells (or a specific subset of them, like these fibroblasts) become more critical as the disease *progresses*.

It also highlights some inconsistencies in the field, possibly due to different mouse models (AngII vs. genetic syndromes) and different ways of tracing cell origins. Science is messy sometimes!

Moving forward, it’s clear we need to:

• Study the transcriptomic differences between origins at later stages of the disease to see how things change over time.
• Find better ways to specifically trace and manipulate CNC-derived cells without causing developmental issues.
• Dive deeper into that specific fibroblast sub-cluster, perhaps using the markers we identified (Ube2c and H2afz), to understand exactly what they’re doing and how they contribute to matrix problems and aortopathy.

Ultimately, understanding these subtle, lineage-specific differences, especially in key cell populations like that fibroblast sub-cluster, could be crucial for developing targeted therapies to prevent or treat this serious condition. It’s complex, but we’re slowly piecing together the puzzle!

Source: Springer