CAPI

Bringing blood back to the body’s extremities Critical limb ischemia is a major health problem across the Western World and with no adequate treatment available, researchers are developing new therapies. A protein called CycloPhilin A is thought to promote angiogenesis in ischemic tissues, and may provide a novel strategy to treat critical limb ischemia, as Dr Patrizia Nigro explains A condition which reduces blood flow to the body’s lower extremities, critical limb ischemia (CLI) is a major public health problem, affecting around 3 million people in Western Europe. Treatment options remain relatively limited however, underlining the importance of continued research into new therapeutic approaches.

“It’s very important to find a therapeutic approach to treat critical limb ischemia, as revascularization is not always feasible,” outlines Dr Patrizia Nigro, the Principal Investigator of the CAPI project. The project aims to explore a new approach to therapy, building on Dr Nigro’s earlier research into the role of a particular protein called CycloPhilin A (CyPA) in cardiovascular disease. “I realised that CyPA could potentially be a proangiogenic molecule, and I am trying to understand the role of this protein in cardiovascular disease,” she continues.

“So I asked: could CyPA be used in a therapeutic approach to orchestrate the mobilisation of progenitor cells, and therefore to increase angiogenesis in ischemic tissue?”

Critical limb ischemia This could form the basis for a novel approach to treat critical limb ischemia, promoting the re-vascularization of affected tissue in patients. This could be a more effective method of treating people with critical limb ischemia than current methods, particularly given that many patients with CLI are elderly and have other co-morbidities, in which case surgery can be risky. “People with diabetes are more likely to get critical limb ischemia, yet the main cause is atherosclerosis.

In atherosclerosis, plaques develop which occlude arteries, including arteries in the lower limbs,” says Dr Nigro. The major problem for

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patients with critical limb ischemia is that they don’t have adequate blood perfusion in the affected limb, so it’s essential to increase angiogenesis – the formation of new blood vessels – in tissue; cell therapy holds clear potential in these terms. “A particular cell population, called the vascular progenitor cells, are involved in critical limb ischemia and can be recruited from the bone marrow to the ischemic limb,” continues Dr Nigro.

involves using an established mouse model to study limb ischemia. In this model the femoral artery is removed and subsequently CyPA is injected. “We found that CyPA, by increasing the recruitment of vascular progenitor cells, induces angiogenesis in these mice,” outlines Dr Nigro. Researchers have also been investigating the underlying processes behind the mobilisation of the vascular progenitor cells. “A receptor

I realised that CyPA could potentially be a pro-angiogenic molecule, and I’ve been trying to understand the role of this protein in cardiovascular disease. So I asked the question; could CyPA be used to increase angiogenesis in ischemic tissue?

By mobilising vascular progenitor cells from the patients’ bone marrow, CyPA could help promote the development of new blood vessels and restore blood flow to the affected limb. “I have demonstrated that CyPA mobilises these progenitor cells, and also increases their proliferation,” explains Dr Nigro.

The project has used two main experimental approaches. The first centres around injecting CyPA directly into the ischemic limb, while the second

called CXCR4 is expressed in progenitor cells, while a factor that binds CXCR4, called stromal derived factor 1 (SDF-1), is released from vascular cells,” continues Dr Nigro. “We’ve found that CyPA enhances the secretion of SDF-1 and via this mechanism attracts CXCR4expressing progenitor cells to the ischemic tissue. However, it is important to point out that angiogenesis involves many cells in our body, not just progenitor cells.”

Left Fig. CyPA increases limb perfusion and angiogenesis.

Right Fig. CyPA enhances BM-derived vascular progenitor cell (CFSE) recruitment in ischemic mice.

EU Research

This research also holds important implications for a number of other conditions. Alongside the project’s research into critical limb ischemia, Dr Nigro also plans to investigate the role of CyPA in other cardiovascular diseases.

“I’m trying to understand the role of CyPA in genetic cardiovascular diseases, in particular in arrhythmogenic cardiomyopathy. We’ve found that CyPA is an important factor in adipose substitution, which we see in patients with arrhythmogenic cardiomyopathy.

CyPA is thought to be critical in determining adipose substitution in people with this type of genetic cardiomyopathy,” she outlines. Improving blood flow could also be an effective treatment method beyond cardiovascular conditions. “Increasing new angiogenesis could be relevant in other contexts, not only in CLI,” acknowledges Dr Nigro.

Marfan syndrome Researchers are also investigating the role of CyPA in Marfan syndrome, a genetic disorder that can lead to complications in many organs, in particular the aorta. Patients with Marfan syndrome are at an increased risk of suffering from an aortic aneurysm; Dr Nigro investigated this area during a period she spent

working in the US. “We found that CyPA was really important for the development of abdominal aortic aneurysms. So we used a mice knock-out for CyPA, and we found that these mice did not develop abdominal aortic aneurysms,” she outlines. Building on this earlier research, Dr Nigro now aims to gain further details about the role of CyPA. “I am currently based in a cardiovascular research hospital, so I have the opportunity to take biopsy materials from human aortic tissue,” she continues. “From these biopsies of patients, I am able to directly examine the tissue or look at cells – in particular vascular smooth muscle cells and fibroblasts from the aorta. I am trying to understand – both in vivo and in vitro – the importance of CyPA in the development of an aneurysm.”

This research is largely fundamental in nature at the moment, with scientists looking to gain further knowledge on the importance of CyPA with respect to critical limb ischemia and several other conditions. However, Dr Nigro is keen to eventually translate the project’s findings into improved treatment of critical limb ischemia. “After using injections of CyPA in the context of critical limb ischemia in a new, improved mouse model, I plan to pursue further research into treating human patients,” she says.

Centro Cardiologico Monzino

At a glance Full Project Title Role of Cyclophilin A in Bone Marrow Vascular Progenitor Cell Mobilization and Recruitment in the Angiogenic Response to Ischemia (CAPI) Project Objectives 1) Characterize CyPA’s effect on isolated PC behavior in vitro.

2) Determine the role of CyPA on PC mobilization from the BM. 3) Evaluate the role of CyPA in circulating PC recruitment into the mouse ischemic hind-limb.

4) Characterize the role of CyPA in new blood vessel development in the mouse ischemic hind-limb.

Translational relevance and impact The execution of the proposed project has the potential to uncover an aspect that is extremely relevant for the human health: the treatment and cure of patients with CLI, a disease with tremendous impact in the world.

Project Funding The CAPI project is funded by the European Research Council.

Contact Details Dr Patrizia Nigro, PhD Unità di Biologia Vascolare e Medicina Rigenerativa Centro Cardiologico Monzino-IRCCS Via Parea 4 20138 Milano, Italia T: +39-02-58002028 E: pnigro@ccfm.it W: https://www.cardiologicomonzino.it/en/

Dr Patrizia Nigro, PhD

Dr Patrizia Nigro, PhD is the Deputy Head of the Unit of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino, Milan, Italy. She has performed ground-breaking research on the role of Cyclophilin A (CyPA) in cardiovascular biology and disease. She contributed unravelling the role of CyPA in vascular remodeling (Circulation, 2008), abdominal aortic aneurysm (Nature Medicine, 2009), atherosclerosis (Journal of Experimental Medicine, 2011) and cardiac hypertrophy (Arteriosclerosis, Thrombosis, and Vascular Biology, 2011). Currently, Dr Nigro is studying the role of CyPA in critical limb ischemia as well as its involvement in cardiovascular aging and genetic cardiomyopathies.

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