Study identifies a surprising new treatment target for chronic limb-threatening ischemia

What causes poor outcomes in patients with advanced peripheral artery disease who develop a complication called chronic limb-threatening ischemia (CLTI), which has a high risk of limb amputation due to the restriction of blood flow to the extremities?

Researchers screened for factors in skeletal muscle samples from patients with CLTI to identify those that were different compared to controls. Surprisingly, it wasn’t growth factors that emerged as different, but a long non-coding RNA (lncRNA) called CARMN—and it wasn’t expressed in endothelial cells, only in vascular smooth muscle cells.

Mark W. Feinberg, MD, cardiologist with the Mass General Brigham Heart and Vascular Institute and professor of medicine at Harvard Medical School, is the senior author of a paper published in the Journal of Clinical Investigation titled “A smooth muscle cell lncRNA controls angiogenesis in chronic limb-threatening ischemia through miR-143-3p/HHIP signaling.” He describes the study in the interview below.

What question were you investigating?

What causes poor outcomes in patients with advanced peripheral artery disease who develop a complication called chronic limb-threatening ischemia (CLTI), which has a high risk of limb amputation due to the restriction of blood flow to the extremities?

For decades, a lot of research into CTLI has focused on understanding endothelial-derived factors—substances released by cells that line our blood vessels—and how those factors lead to the growth of new blood vessels. (The development of new blood vessel from existing ones is called angiogenesis.) The idea is that if we can find a therapy that helps patients with CLTI produce more blood vessels, we can improve blood flow to threatened limbs and reduce the risk of amputation or other health complications.

To date, the growth factors those studies have identified have failed in clinical trials to improve outcomes. Our study points to a different approach. We screened for factors in skeletal muscle samples from patients with CLTI to identify those that were different compared to controls.

Surprisingly, it wasn’t growth factors that emerged as different, but a long non-coding RNA (lncRNA) called CARMN—and it wasn’t expressed in endothelial cells, only in vascular smooth muscle cells.

What methods or approach did you use?

We used a range of transcriptomic profiling approaches to identify the lncRNA CARMN in human skeletal muscle biopsies and in mouse models of limb ischemia.

We developed a knockout mouse of the lncRNA CARMN which exhibited impaired blood flow recovery, limb necrosis, and amputation in a similar manner to CLTI patients that have reduced expression levels of this lncRNA in skeletal muscle biopsies.

What did you find?

We found that a unique protein called HHIP, made by smooth muscle cells, is controlled by lncRNA CARMN. HHIP helps manage blood vessel growth, blood flow, and tissue healing.

When HHIP was blocked—or when another molecule that controls HHIP was increased—blood vessels grew better, and damaged tissue healed more effectively. This reveals a new way that smooth muscle cells and blood vessel cells work together, which scientists hadn’t understood before.

What was surprising about your study?

Despite this lncRNA not being expressed in endothelial cells that make capillaries, mice that can’t produce this lncRNA have reduced capillaries in their skeletal muscles with limb ischemia. HHIP appears to be the missing link, connecting what’s happening in smooth muscle cells (SMCs) to the effects we see in endothelial cells (ECs). Inhibition of HHIP or overexpression of a microRNA that regulates HHIP was sufficient to fully rescue angiogenesis, limb tissue perfusion, and repair.

What are the implications?

The work provides new therapeutic strategies for chronic limb-threatening ischemia and provides new insights into SMC-EC crosstalk that was not previously understood in the field of angiogenesis.

What are the next steps?

We’re trying to figure out why the molecule CARMN drops when blood flow is blocked in the limbs. We’ve found a promising new target that may control CARMN when oxygen levels are low. This could lead to new ways to boost CARMN, improve blood flow, and help heal tissues—potentially benefiting people with various heart and blood vessel problems such as peripheral artery disease and CLTI.