Skip to main content



Critical limb ischemia (CLI) is a major health problem; it affects approximately three million people in Western Europe, its prevalence is increasing and adequate medical treatment is not available for patients with advanced disease. Since CLI is characterized by inadequate tissue vascularisation, much effort has been focused on strategies aimed at promoting neoangiogenesis. Emerging data suggest the clinical potential of vascular progenitor cells (PC) to restore blood flow to the ischemic limb. Several factors have been shown to influence PC mobilization and homing to ischemic tissue. However, follow-up studies have yet to show long-term benefit. Therefore, the identification of novel molecules represents a major and clinically relevant task. One factor that mediates the cross-talk between PC and a variety of cell types, including vascular cells, is stromal-derived factor 1 (SDF-1), which, through interaction with its receptor CXCR4 expressed on PC, modulates the role of PC in angiogenesis. A key mediator of SDF-1/CXCR4 axis is Cyclophilin A (CyPA). Specifically, CyPA binds to CXCR4 and modulates SDF-1-mediated chemotactic cell migration. Furthermore, CyPA has been shown to enhance endothelial cell (EC) proliferation as well as capillary-like structure development and migration. Importantly, our published data show that CyPA is crucial for the mobilization and recruitment of bone marrow (BM)-derived cells in the diseased aorta of ApoE-/- mice. However, the role of CyPA in PC function is unknown. Based on experimental evidences underlying the importance of CyPA in SDF-1/CXCR4 axis, ability to modulate EC function in vitro, and BM cell mobilization or recruitment, this proposal aimed to evaluate the role of CyPA in the regulation of PC and new blood vessel development in response to ischemia.

To reach this goal we have proposed the following aims:

Aim 1: Characterize CyPA’s effect on isolated PC behavior in vitro.
To examine the effect of extracellular CyPA on PC, we isolated these cells from the BM of wild-type (WT) mice and treated them with different concentrations of exogenously administered CyPA (recombinant protein) in vitro. Low concentrations (100 ng/ml) of CyPA stimulated PC proliferation while higher concentrations (1 μg/ml) promoted cell death. Furthermore, the effect of CyPA on the adhesive and migratory activity of PC was investigated. CyPA enhanced cell adhesion to fibronectin-coated dishes in a dose-dependent-manner and directly stimulated PC migration.
In order to evaluate the role of endogenous CyPA on PC behaviour, we isolated PC from CyPA knockout mice (CyPA-/-) and we found that these cells migrated significantly less than WT cells in response to SDF-1 treatment.

Aim 2: Determine the role of CyPA on PC mobilization from the BM.
Emerging data suggest the importance of PC mobilization in the process of revascularization following ischemia.
We found that exogenous administered CyPA increases the mobilization of PC from the BM to the peripheral circulation after acute hind-limb ischemia (HLI). This effect was significantly reduced in CyPA-/- mice compared to WT mice. In light of the major importance of the SDF-1/CXCR4 axis on PC mobilization from the BM into the bloodstream, we evaluated the effect of AMD3100, a CXCR4 antagonist, in CyPA-induced PC mobilization. Interestingly, AMD3100 treatment in the presence of CyPA significantly decreased PC mobilization. These results suggest that CyPA is an effective chemoattractant factor after ischemic injury and it acts by regulating the SDF-1/CXCR4 axis.

Aim 3: Evaluate the role of CyPA in circulating PC recruitment into the mouse ischemic hind-limb.
After mobilization from the BM, PC are recruited to the site of ischemia where they participate in functional repair. We found that exogenously administered CyPA also increases the recruitment of PC into the ischemic tissue and this effect was significantly decreased in CyPA-/- mice.
Mechanistic studies revealed that CyPA increases SDF-1 serum levels and up-regulates CXCR4 in PC and in adductor muscles after ischemia. Additionally, SDF-1/CXCR4 axis inhibition by the CXCR4 antagonist AMD3100 decreased CyPA-mediated PC recruitment in the ischemic limb. These data suggest that CyPA modulates PC recruitment into ischemic tissue by regulating SDF-1/CXCR4 pathway.

Aim 4: Characterize the role of CyPA in new blood vessel development in the mouse ischemic hind-limb.
In order to provide further confirmation for the functional role of CyPA in post-injury neovascularization, we found that limb perfusion, capillary density, and arteriole length density were significantly increased after CyPA treatment. Consistently, genetic ablation of CyPA expression in CyPA-/- mice caused a significant impairment of neovascularization in ischemic areas.

Collectively these data suggest that CyPA can play a crucial role in enhancing SDF-1/CXCR4 axis activation. This mechanism is important to increase the recruitment of BM-derived PC into the ischemic site, leading to an improved neo-angiogenic process. Thus, CyPA may serve as a useful therapeutic strategy for accelerating angiogenesis in ischemic cardiovascular diseases, such as CLI.

Note that the most relevant findings are illustrated in our recently published article (see attachment).

Related documents