Retention of endothelial progenitor cells in bone marrow in a murine model of endogenous tissue plasminogen activator (tPA) deficiency in response to critical limb ischemia

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Abstract

Background

This study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating the left femoral artery.

Methods

Wild-type (C57BL/6) (n = 40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0 mg/kg)] and group 4A [CLI-tPA (intravenously at 3 h after CLI)]. Similarly, tPA knock-out (tPA−/−) mice (n = 40) were equally divided into group 1B (sham control), group 2B (CLI), group 3B [control-tPA (4.0 mg/kg)], and group 4B (CLI-tPA).

Results

The circulating levels of EPCs (C-kit/CD31 +, Sca-1/KDR +, CXCR4/CD34 +) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p < 0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p > 0.05) at 6 h and 18 h post-CLI. Levels of these biomarkers decreased again 14 days after CLI in tPA−/− mice compared to those in wild-type between the respective groups (all p < 0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p < 0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4 +, SDF-1α +, and CD31 + cells) levels was highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA−/− mice (p < 0.01).

Conclusion

tPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model.

Introduction

Studies have previously shown that mobilization of endothelial progenitor cell (EPC) from bone marrow to circulation and migration to ischemic area for angiogenesis/vasculogenesis through an up-regulation of matrix metalloproteinase (MMP)-9 [1] and down-regulation of CD26/Dipeptidylpeptidase IV (DPP IV) system [2] are essential for the improvement of ischemic organ dysfunction [1], [2]. Intriguingly, these studies [1], [2] consistently emphasized that the mobilization of EPCs from bone marrow (BM) to circulation only occurs in the setting of tissue ischemia. The mechanisms involved in the regulation of the kinetics of EPC mobilization, however, have not been elucidated [1], [2].

On the other hand, previous studies have revealed that membrane-bound molecules play an essential role in the adhesion of EPCs to BM-stromal cells [3], [4], [5]. For instance, BM-stromal cells express membrane-bound C-kit ligand (C-kit-L) which binds to the membrane receptor C-kit of EPCs. MMP-9 acts by cleaving the membrane-bound C-kit-L to the soluble form C-kit-L, which then interacts with the EPC C-kit receptor to initiate the signal (i.e., the downstream signaling of MMP-9) that is crucial for BM-EPC differentiation and mobilization to systemic circulation [3], [4], [5], [6]. Certainly, to further understand the possible mechanisms by which MMP-9 participates in the mobilization of bone marrow EPCs to circulation, not only the downstream signaling pathway but also the upstream one should be clarified. In fact, the upstream signaling pathway that mediates MMP-9 activity has not been reported. Based on the common concept that atherothrombotic ischemia or acute vessel occlusion caused by thrombus formation activates tissue plasminogen activator (tPA), we have recently investigated the possible role of tPA in the regulation of MMP-9 activity using a murine model of critical limb ischemia (CLI) [7]. Our results showed that tPA treatment enhanced the kinetics and circulating level of EPCs, angiogenesis, and blood flow in the ischemic limb through upregulating MMP-9 activity in BM and SDF-1α concentration in circulation [7]. Additionally, in a sciatic nerve and spinal injury animal model, studies have previously demonstrated that tPA deficiency attenuated MMP-9 activity and macrophage migratory ability, thereby delaying axon regeneration [8], [9]. Hence, in addition to promoting reperfusion in the setting of acute ST-segment elevation myocardial infarction [10], [11], tPA may be involved in other critical physiological processes through modulating inter-cellular communication, extracellular matrix interaction, or intracellular signal transduction [7], [8], [9], [12].

However, the limitation of our recent study [7] is that, without using a tPA knock-out (tPA−/−) animal model, the role of endogenous tPA in the upstream signaling pathway remains unclear. In fact, systemic administration of tPA in our study [7] may only partially reflect the effects of tPA that are also transient and mainly confined to the peripheral circulation rather than equal distribution in each organ. To directly clarify the role of endogenous tPA in the recovery from ischemic insults and its involvement in vivo as well as to evaluate whether exogenous tPA can reverse the tPA deficiency-related reduction in circulating EPC level and enhance angiogenesis, we compared the tPA-deficient (tPA−/−) mice and wild-type mice with a common genetic background in an attempt to elucidate the role of tPA in regulating the activity of MMP-9 and its impact on EPC mobilization and angiogenesis in a CLI setting.

Section snippets

Ethics

All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital and performed (Affidavit of Approval of Animal Use Protocol No. 2010032201) in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, National Academy Press, Washington, DC, USA, revised 1996).

Animal model of critical limb ischemia and experimental protocol

Ten-week-old male wild-type C57BL/6 mice (n = 40), weighing 22–23 g, (Charles River Technology, BioLASCO Taiwan Co., Ltd.,

Flow cytometric quantification of serial changes in circulating level of c-Kit/CD31 + cells

At 6 h (Fig. 1A) and 18 h (Fig. 1B) after the CLI procedure, the percentage of circulating c-Kit/CD31 + cells was significantly higher in wild-type (group 1A) than that in tPA−/− (group 1B) normal controls, and significantly higher in the wild-type CLI group (group 2A) than in the tPA−/− CLI group (group 2B). However, the percentage did not differ between wild-type + tPA group (group 3A) and tPA−/− + tPA group (group 3B) or between the wild-type CLI + tPA (group 4A) and tPA−/− CLI + tPA (group 4B) groups.

Discussion

Using a tPA−/− mouse model of CLI, this study investigated the distinctive role of tPA in enhancing EPC mobilization from BM to circulation and recruitment of EPCs in ischemic tissue through regulating MMP-9 activity and yielded several striking implications. First, compared with the wild-type mice, the baseline circulating EPC level was notably lower in the tPA−/− mice and was reversed at 6 h (Fig. 1A) and 18 h (Fig. 1B) after a single dose of tPA in group 3B. However, the difference in

Study limitations

This study has limitations. This study was designed to imitate the routine clinical administration of one dose of tPA to patients with STEMI or acute ischemic stroke. Accordingly, whether repeated dosage or over-expression of tPA would further upregulate the circulating level of EPCs and improve the blood flow in this murine CLI model remains uncertain.

Conclusions

In conclusion, the retention of EPCs in the BM niche in a murine model of endogenous tPA deficiency at baseline and after CLI induction could be transiently reversed in the acute phase through exogenous tPA replacement. Not only did tPA treatment promote EPC mobilization into the circulation and ischemic muscle, but also it enhanced angiogenesis and blood flow. These findings may highlight the novel concept of modifying the routine clinical dosage of tPA to achieve beneficial biological effect

Acknowledgments

This study was supported by a program grant from the National Science Council, Taiwan, R.O.C. (Grant number: NSC-99-2314-B-182A-093-MY3). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.

References (18)

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1

Indicates equal contribution in this study compared with the first author.

2

Indicates equal contribution in this study compared with the corresponding author.

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