Brief report: Mechanism of extravasation of infused stem cells

Abstract

In order for bloodborne stem cells to be effective in tissue regeneration, cells must cross vessel walls and enter the parenchyma. Although such transmigration does occur, the mechanism remains elusive. Leukocytes invade tissue by diapedesis; stem cells are commonly assumed to do likewise, but evidence is lacking. Cardiac-derived regenerative cells and multicellular cardiospheres (CSPs) were infused into the coronary vessels of rat hearts. Serial histology revealed a novel mechanism of cell transmigration, "active vascular expulsion," which underlies the extravasation of infused cells and cell aggregates. In this mechanism, the vascular barrier undergoes extensive remodeling, while the cells themselves are relatively passive. The mechanism was confirmed in vivo by serial intravital microscopy of CSP extravasation in a dorsal skin flap model. Integrins and matrix metalloproteinases play critical roles in active vascular expulsion. In vitro models revealed that active vascular expulsion is generalizable to other stem cell types and to breast cancer cells. Recognition of active vascular expulsion as a mechanism for transvascular cell migration opens new opportunities to enhance the efficacy of vascularly delivered cell therapy.

Figure 1. Endothelial pocketing and vascular expulsion leading to extravasation of cardioshere-derived cells (CDCs) and cardiospheres (CSPs)

a & b, Confocal imaging shows the membrane projections from adjacent endothelium (white arrowheads) surrounding a CDC (a; green) and CSP (b; green) 24 hours after infusion. The original vascular wall undergoes breakdown to create a path for CDC and CSP extravasation (white arrows; a & b). Restoration of vessel patency is evident from circulating blood cell nuclei in the newly-recanalized lumen (b; yellow arrows). c, Beginning of endothelial pockets via endothelial cell projection (white arrowheads). d, Single-time-point confocal imaging of heart sections shows the extravasation time courses of CSP and CDC, and the lack of extravasation with PSP. e, Occlusion of a microvessel by a CSP or a PSP (green) induced focal hypoxia as detected by Hypoxyprobe^™ (magenta). f, CSP and CDC extravasate within 72 hours of infusion (mean ± s.d.; n = 3 rats per time point). PSP did not undergo extravasation. *, P < 0.05 when compared to PSP. g, Vessel patency was re-established in the CSP and CDC groups but not in the PSP group. *, P < 0.05 when compared to PSP. h, Quantification of Hypoxyprobe^™ fluorescence in CSP- and PSP-injected rats (mean ± s.d.; n = 3 rats per time point; asterisk, P < 0.05, two-tailed Student’s t-test). Scar bars = 20 μm in a, b, c & d and 100 μm in e.

Figure 2. Live imaging of active vascular expulsion in a mouse skin flap model

a, steps to create dorsal skin flap and perform intravital confocal imaging. b, extravasation of a CDC by sequential active vascular expulsion process. New vascular walls were formed by endothelial projections (white arrows) and the lumen was patent. The opposing wall of the endothelial pocket was broken down (white arrow head) to allow the CDC to be expelled into the extravascular space. Bars = 5 μm. c, an intact CSP (yellow arrow) were found in the extravascular space 72 hours after injection. The CDCs (white arrows) could be originated from CSP dissociation after extravasation. Bar = 50 μm.

Figure 3. Integrins are required for the initiation of endothelial pocketing

a & b, Endothelial pocketing was recreated in vitro when CSP (green) were plated upon monolayers of human vein endothelial cells (HUVEC; red). White asterisks show endothelial pockets formed by HUVEC surrounding CSPs. Blue marks cell nuclei (DAPI). c, Plain PSP did not elicit responses from the HUVEC but fibronectin-coated PSP induced HUVEC projections; CSP were surrounded by HUVEC projections, but not when incubated with RGDS peptide. *, P < 0.001 when compared to PSP. **, P < 0.05 when compared to all other groups. d & e, Similar HUVEC projections occurred in vitro with cellular spheres created from other cell types: mesenchymal stem cells (d) and MCF-7 breast cancer cells (e). f, Quantification of CSP retention within tissue 10 min after infusion in rats treated with integrin-blocking peptide (RGDS) or the scrambled version (Control). RGDS reduced initial adhesion of CSP to the blood vessels. Blocking integrins prevented the formation of endothelial pockets 24 hours after infusion (g), leading to suppression of extravasation (h) and potentiation of tissue hypoxia (i) (n = 5 rats per group;*, P < 0.05 when compared to Control). Scar bars = 50 μm.

Figure 4. MMPs are required for pocket breakdown and active expulsion

a, Confocal imaging shows MMPs (magenta) concentrate in the vascular wall around a CSP (green). Scale bar, 50 μm. b, Quantification of MMP2 fluorescence in CSP- and PSP-injected rats. MMPs increase markedly at 24 hr, then decline, in response to CSP infusion; in contrast, MMPs are activated by PSP weakly and slowly (asterisk, P < 0.01; n = 3 rats per time point). c, The extravasation of CSP 72 hours after infusion is largely blocked by the broad-spectrum MMP inhibitor GM6001, but not by a negative control compound (GM6001-NC) (*, P < 0.01; n = 5 rats per group). d, Blocking of MMPs does not affect endothelial pocketing 24 hours after infusion (P = 0.77; n = 5 rats per group).

Figure 5. Simulation of CSP extravastion in vitro

a, Confocal imaging shows a CSP which landed far from the endothelial tubes and remains isolated. b, CSP which happened to fall upon a tubular nexus were quickly surrounded by HUVEC projections. c, 3D-reconstructed confocal imaging of the CSP and HUVEC in (b). The cartoon indicates the process whereby the CSP is pocketed by HUVEC projections and then penetrates into the underlying Matrigel. Scale bars = 50 μm. d, Time course of endothelial pocketing and CSP penetration in vitro. e, CSP penetration 48 hr after plating was blocked by the broad-spectrum MMP inhibitor GM6001, but not by the negative control compound (GM6001-NC) (*, P < 0.001). f, Blocking of MMPs does not affect formation of endothelial pockets in vitro (P = 0.69). Experiments were performed in triplicates.

Figure 6. Integrative implications of active vascular expulsion

a, Masson trichrome staining of heart sections at 3 weeks shows myocardial infarction (blue) in PSP-infused rats, but not in CSP- or Saline-infused rats. Scale bar, 5 mm. b, Quantification of infarct size in CSP-, PSP-, and saline-infused rats (n = 5 rats per group; asterisk, P < 0.01 compared to Saline). c, Serum troponin I indicates micro-embolic injury in PSP-infused animals, but not in CSP-infused ones (n = 3 rats per group; *, P < 0.01 compared to Saline). d, Echocardiography revealed normal cardiac function (left ventricle ejection fraction; LVEF) in Saline- and CSP-infused animals at 3 weeks. LVEF deteriorated in the PSP-infused animals over the 3 week time course, consistent with the induction of myocardial infarction by PSP infusion in that group only (n=5–6 rats per group; *, P < 0.05 compared to Saline). e, Schematic depiction of steps in active vascular expulsion.