Role of kinin B2 receptor signaling in the recruitment of circulating progenitor cells with neovascularization potential

Abstract

Reduced migratory function of circulating angiogenic progenitor cells (CPCs) has been associated with impaired neovascularization in patients with cardiovascular disease (CVD). Previous findings underline the role of the kallikrein-kinin system in angiogenesis. We now demonstrate the involvement of the kinin B2 receptor (B(2)R) in the recruitment of CPCs to sites of ischemia and in their proangiogenic action. In healthy subjects, B(2)R was abundantly present on CD133(+) and CD34(+) CPCs as well as cultured endothelial progenitor cells (EPCs) derived from blood mononuclear cells (MNCs), whereas kinin B1 receptor expression was barely detectable. In transwell migration assays, bradykinin (BK) exerts a potent chemoattractant activity on CD133(+) and CD34(+) CPCs and EPCs via a B(2)R/phosphoinositide 3-kinase/eNOS-mediated mechanism. Migration toward BK was able to attract an MNC subpopulation enriched in CPCs with in vitro proangiogenic activity, as assessed by Matrigel assay. CPCs from cardiovascular disease patients showed low B(2)R levels and decreased migratory capacity toward BK. When injected systemically into wild-type mice with unilateral limb ischemia, bone marrow MNCs from syngenic B(2)R-deficient mice resulted in reduced homing of sca-1(+) and cKit(+)flk1(+) progenitors to ischemic muscles, impaired reparative neovascularization, and delayed perfusion recovery as compared with wild-type MNCs. Similarly, blockade of the B(2)R by systemic administration of icatibant prevented the beneficial effect of bone marrow MNC transplantation. BK-induced migration represents a novel mechanism mediating homing of circulating angiogenic progenitors. Reduction of BK sensitivity in progenitor cells from cardiovascular disease patients might contribute to impaired neovascularization after ischemic complications.

Figure 1

Flow cytometry shows the expression of kinin receptors on freshly isolated PB-MNCs, CD133⁺, and CD34⁺ progenitors, as well as cultured EPCs of healthy human subjects. A, B₂R is highly abundant in progenitor CD133⁺ and CD34⁺ cell fractions as compared with total MNCs, whereas B₁R expression is low in the studied cell populations. B and C, Reverse gating shows that in the B₂R⁺ MNC fraction, CD133⁺ and CD34⁺ progenitors (B) coexpressing KDR and CXCR4 (C) are more abundant. D, Consistently, outgrowth of acLDL⁺UEAI⁺ EPCs is more frequent from FACS sorted B₂R⁺ PB-MNCs. Values are means±SEM; n=6 to 15. **P<0.01.

Figure 2

A and B, In human EPCs, BK induces B₂R-dependent filopodia formation (n=6) (arrows) (A) and polarization of rac1 (n=9) (arrow) (B) as compared with unstimulated control (con). B, Right, Isotype FITC staining. DiI was used as a nonpolarizing label in B. Scale bars, 5 μm. C and F, In transwell assays, directed migration of EPCs toward BK (n=16) was abolished in the presence of the B₂R antagonist icatibant (ic) (n=10), PI3K inhibitor LY294002 (LY) (n=6), and eNOS inhibitor L-NIO (n=4) (C) and after silencing of rac1 (n=4) (F). D, Scratch assay demonstrated an increased B₂R-dependent motility of BK-stimulated human EPCs (n=10). E, Migration of murine BM-EPCs toward BK was B₂R-dependent, being inhibitable by icatibant (WT EPCs) and abrogated by B₂R gene deletion (B₂R^−/− EPCs) but not by B₁R gene deletion (B₂R^−/− EPCs) (n=6 for all). Values are means±SEM. *P<0.05, **P<0.01.

Figure 3

A, CD133⁺ and CD34⁺ progenitor cells coexpressing KDR or CXCR4 are enriched in the BK^mig population. B, BK^mig PB-MNCs give rise to more acLDL⁺UEAI⁺ EPCs in vitro as compared with BK^non. EPCs derived from BK^mig or BK^non PB-MNCs represent a heterogeneous population harboring elongated cells that integrate into HUVEC networks (#), as well as rounded cells that do not incorporate but stay in proximity to developing networks (§). C, Both populations might modulate network formation. Scale bar, 10 μm). D through H, BK^mig-derived EPCs support in vitro network formation of HUVECs more than BK^non-derived EPCs (n=6) (scale bars, 100 μm) (D through G) and secrete more proangiogenic IL-8 and MCP-1 and less IL-1β and RANTES (n=6) (H). Values are means±SEM. *P<0.05, **P<0.01.

Figure 4

A through C, B₂R is coexpressed on a lower number of circulating CD34⁺ (A) and CD133⁺ progenitor cells (B) of patients with SA (n=23) and aMI (n=27), as well as on a lower number of cultured EPCs from SA patients (n=8) (C), as compared with healthy controls (n=32). The capacity of BK migration assay to enrich for CD34⁺ (D) and CD133⁺ (E) progenitor cells from PB-MNCs was evaluated in patients with SA or aMI as compared with healthy subjects (n=8 per group). E and F, Results indicate abrogation of BK responsiveness in all studied populations of SA patients, as documented by a similar enrichment ratio in BK stimulated or unstimulated (con) cells, whereas BK sensitivity was conserved in CD133⁺ cells coexpressing KDR or CXCR4 of aMI patients. G, Furthermore, BK^mig MNCs from aMI patients and healthy donors support network formation by HUVECs more than BK^non, whereas BK^mig from SA patients are ineffective and not superior to BK^non. Values are means±SEM or expressed as box and whiskers. *P<0.05, ***P<0.005.

Figure 5

A and B, Network formation by HUVECs is supported by coculture with WT BM-EPCs (A) but not with or B₂R^−/− BM-EPCs (B). Scale bars, 100 μm. C and D, Bar graphs show average thickness (C) and cumulative length of the branches (D) (n=8 samples per group).

Figure 6

A, Homing of total, sca1⁺, or cKit⁺ flk1⁺ BM-MNCs from the circulation to the ischemic adductor of WT recipients is reduced when B₂R^−/− BM-MNCs are injected or under systemic blockade of the B₂R by icatibant (n=8 per group). B, This remains evident when normalizing to the number of donor cells (total MNCs) persistent in the recipient blood. C and D, Blood flow recovery measured in the paw by Doppler flowmetry (**P<0.01, ++ P<0.01 WT vs vehicle and B₂R^−/−, respectively) (C) and in the adductor by fluorescent microspheres (*P<0.05) (D) was less efficient in mice receiving vehicle or B₂R^−/− BM-MNCs as compared with WT BM-MNCs (n=8 per group). E and F, In ischemic adductors of mice receiving B₂R^−/− BM-MNCs, both capillary density (*P<0.05, ***P<0.005) (E) and vessel area, as normalized per muscle area (*P<0.05) (F), were reduced. Values are means±SEM or expressed as box plot.