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Cathepsin L is required for endothelial progenitor cell–induced neovascularization

Abstract

Infusion of endothelial progenitor cells (EPC), but not of mature endothelial cells, promotes neovascularization after ischemia. We performed gene expression profiling of EPC and endothelial cells to identify genes that might be important for the neovascularization capacity of EPC. Notably, the protease cathepsin L (CathL) was highly expressed in EPC as opposed to endothelial cells and was essential for matrix degradation and invasion by EPC in vitro. CathL-deficient mice showed impaired functional recovery following hind limb ischemia, supporting the concept of a crucial role for CathL in postnatal neovascularization. Infused CathL-deficient progenitor cells neither homed to sites of ischemia nor augmented neovascularization. Forced expression of CathL in mature endothelial cells considerably enhanced their invasive activity and sufficed to confer their capacity for neovascularization in vivo. We concluded that CathL has a critical role in the integration of circulating EPC into ischemic tissue and is required for EPC-mediated neovascularization.

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Figure 1: Gene expression analysis of EPC and HUVEC.
Figure 2: The expression and activity of cathepsin L is increased in EPC.
Figure 3: Role of CathL in neovascularization.
Figure 4: Role of CathL in EPC invasion.
Figure 5: CathL is required for improvement of neovascularization.
Figure 6: Overexpression of CathL in mature endothelial cells partially rescued the impaired improvement of neovascularization.

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References

  1. Isner, J.M. & Asahara, T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J. Clin. Invest. 103, 1231–1236 (1999).

    Article  CAS  Google Scholar 

  2. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1, 27–31 (1995).

    Article  CAS  Google Scholar 

  3. Risau, W. Mechanisms of angiogenesis. Nature 386, 671–674 (1997).

    Article  CAS  Google Scholar 

  4. Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).

    Article  CAS  Google Scholar 

  5. Asahara, T. et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964–967 (1997).

    Article  CAS  Google Scholar 

  6. Shi, Q. et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 92, 362–367 (1998).

    CAS  PubMed  Google Scholar 

  7. Kalka, C. et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA 97, 3422–3427 (2000).

    Article  CAS  Google Scholar 

  8. Kawamoto, A. et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 103, 634–647 (2001).

    Article  CAS  Google Scholar 

  9. Assmus, B. et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 106, 3009–3017 (2002).

    Article  Google Scholar 

  10. Kocher, A.A. et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7, 430–436 (2001).

    Article  CAS  Google Scholar 

  11. Joyce, J.A. et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell 5, 443–453 (2004).

    Article  CAS  Google Scholar 

  12. Berchem, G. et al. Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis. Oncogene 21, 5951–5955 (2002).

    Article  CAS  Google Scholar 

  13. Krueger, S., Kellner, U., Buehling, F. & Roessner, A. Cathepsin L antisense oligonucleotides in a human osteosarcoma cell line: effects on the invasive phenotype. Cancer Gene Ther. 8, 522–528 (2001).

    Article  CAS  Google Scholar 

  14. Dimmeler, S. et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J. Clin. Invest. 108, 391–397. (2001).

    Article  CAS  Google Scholar 

  15. Vasa, M. et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ. Res. 89, E1–7 (2001).

    Article  Google Scholar 

  16. Urbich, C. et al. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation 108, 2511–2516 (2003).

    Article  Google Scholar 

  17. Fiebiger, E. et al. Invariant chain controls the activity of extracellular cathepsin L. J. Exp. Med. 196, 1263–1269 (2002).

    Article  CAS  Google Scholar 

  18. Walker, B., Lynas, J.F., Meighan, M.A. & Bromme, D. Evaluation of dipeptide alpha-keto-beta-aldehydes as new inhibitors of cathepsin S. Biochem. Biophys. Res. Commun. 275, 401–405 (2000).

    Article  CAS  Google Scholar 

  19. Li, Z. et al. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J. Biol. Chem. 279, 5470–5479 (2004).

    Article  CAS  Google Scholar 

  20. Chauhan, S.S., Ray, D., Kane, S.E., Willingham, M.C. & Gottesman, M.M. Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L. Biochemistry 37, 8584–8594 (1998).

    Article  CAS  Google Scholar 

  21. Libby, P. & Schonbeck, U. Drilling for oxygen: angiogenesis involves proteolysis of the extracellular matrix. Circ. Res. 89, 195–197 (2001).

    Article  CAS  Google Scholar 

  22. Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389–395 (2000).

    Article  CAS  Google Scholar 

  23. Devy, L. et al. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002).

    Article  CAS  Google Scholar 

  24. Rooprai, H.K. & McCormick, D. Proteases and their inhibitors in human brain tumours: a review. Anticancer Res. 17, 4151–4162 (1997).

    CAS  PubMed  Google Scholar 

  25. Turk, V., Turk, B. & Turk, D. Lysosomal cysteine proteases: facts and opportunities. EMBO J. 20, 4629–4633 (2001).

    Article  CAS  Google Scholar 

  26. Stypmann, J. et al. Dilated cardiomyopathy in mice deficient for the lysosomal cysteine peptidase cathepsin L. Proc. Natl. Acad. Sci. USA 99, 6234–6239 (2002).

    Article  CAS  Google Scholar 

  27. Tobin, D.J. et al. The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling. Am. J. Pathol. 160, 1807–1821 (2002).

    Article  CAS  Google Scholar 

  28. Shi, G.P. et al. Deficiency of the cysteine protease cathepsin S impairs microvessel growth. Circ. Res. 92, 493–500 (2003).

    Article  CAS  Google Scholar 

  29. Lyden, D. et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat. Med. 7, 1194–1201 (2001).

    Article  CAS  Google Scholar 

  30. Rafii, S., Lyden, D., Benezra, R., Hattori, K. & Heissig, B. Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat. Rev. Cancer 2, 826–835 (2002).

    Article  CAS  Google Scholar 

  31. Nakagawa, T. et al. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science 280, 450–453 (1998).

    Article  CAS  Google Scholar 

  32. Roth, W. et al. Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling. FASEB J. 14, 2075–2086 (2000).

    Article  CAS  Google Scholar 

  33. Houseweart, M.K. et al. Cathepsin B but not cathepsins L or S contributes to the pathogenesis of Unverricht-Lundborg progressive myoclonus epilepsy (EPM1). J. Neurobiol. 56, 315–327 (2003).

    Article  CAS  Google Scholar 

  34. Saftig, P. et al. Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells. EMBO J. 14, 3599–3608 (1995).

    Article  CAS  Google Scholar 

  35. Lelongt, B. et al. Matrix metalloproteinase 9 protects mice from anti-glomerular basement membrane nephritis through its fibrinolytic activity. J. Exp. Med. 193, 793–802 (2001).

    Article  CAS  Google Scholar 

  36. Urbich, C. et al. Dephosphorylation of endothelial nitric oxide synthase contributes to the anti-angiogenic effects of endostatin. FASEB J. 16, 706–708 (2002).

    Article  CAS  Google Scholar 

  37. Premzl, A., Zavasnik-Bergant, V., Turk, V. & Kos, J. Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro. Exp. Cell. Res. 283, 206–214 (2003).

    Article  CAS  Google Scholar 

  38. Aicher, A. et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat. Med. 9, 1370–1376 (2003).

    Article  CAS  Google Scholar 

  39. Dennhofer, R. et al. Invasion of melanoma cells into dermal connective tissue in vitro: evidence for an important role of cysteine proteases. Int. J. Cancer 106, 316–323 (2003).

    Article  Google Scholar 

  40. Kaberdin, V.R. & McDowall, K.J. Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix. Genome Res. 13, 1961–1965 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Hinds, K.A. et al. Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood 102, 867–872 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We are thankful to J. Gille (University of Frankfurt, Frankfurt, Germany) and Z. Werb (University of California, San Francisco, California) for providing the MMP9-deficient mice. DCG-04 was donated by M. Bogyo (Stanford University Medical School, California). We would like to thank A. Knau, M. Näher, and M. Muhly-Reinholz for technical help. This study was supported by the Deutsche Forschungsgemeinschaft (Di 600/4-1, He 3044/2-2 and VA151/4-3), the Alfried Krupp Stiftung (S.D.), the Fonds der Chemischen Industrie (T.R. and C.P.), by the Deutsche Krebshilfe (T.R.), and in part by National Institutes of Health Grant HL071954A (DE-AC03-76SF00098 (L.A.P.)). K.S was in part supported by the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. The authors C.U., C.H., A.A., T.B., A.M.Z. and S.D. belong to the European Vascular Genomics Network (http://www.evgn.org) a Network of Excellence supported by the European Community's sixth Framework Programme for Research Priority 1 “Life sciences, genomics and biotechnology for health” (Contract number LSHM-CT-2003-503254).

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Correspondence to Stefanie Dimmeler.

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Supplementary information

Supplementary Fig. 1

Effect of lymphocyte infusion on neovascularization. (PDF 126 kb)

Supplementary Fig. 2

Expression of cathepsin L on different cell types. (PDF 36 kb)

Supplementary Fig. 3

Effect of cathepsin L on progenitor cell function. (PDF 23 kb)

Supplementary Fig. 4

Role of cathepsin L in mature endothelial cell function. (PDF 45 kb)

Supplementary Fig. 5

Proteolytic activity of EPC and progenitor cells in lysates and supernatants. (PDF 25 kb)

Supplementary Fig. 6

Confocal proteolysis assay. (PDF 73 kb)

Supplementary Fig. 7

Histological evaluation of tissue vascularization—capillary density. (PDF 317 kb)

Supplementary Fig. 8

Histological evaluation of tissue vascularization—conductance vessels. (PDF 59 kb)

Supplementary Fig. 9

Role of cathepsin L in progenitor cell–induced perfusion. (PDF 296 kb)

Supplementary Fig. 10

Incorporation of endothelial progenitor cells. (PDF 101 kb)

Supplementary Fig. 11

Role of cathepsin L in tumor vascularization. (PDF 417 kb)

Supplementary Fig. 12

Role of cathepsin L in incorporation of bone marrow cells. (PDF 63 kb)

Supplementary Fig. 13

MMP-9 and cathepsin L activity in ischemic tissues. (PDF 24 kb)

Supplementary Table 1

The mRNA expression (normalized data) of various proteases and protease inhibitors in EPC, HUVEC and monocytes is summarized. n = 3; *P < 0.05 versus HUVEC. (PDF 19 kb)

Supplementary Movie 1

Incorporation of endothelial progenitor cells (AVI 15480 kb)

Supplementary Methods (PDF 49 kb)

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Urbich, C., Heeschen, C., Aicher, A. et al. Cathepsin L is required for endothelial progenitor cell–induced neovascularization. Nat Med 11, 206–213 (2005). https://doi.org/10.1038/nm1182

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