Juxtacrine and paracrine interactions of rat marrow-derived mesenchymal stem cells, muscle-derived satellite cells, and neonatal cardiomyocytes with endothelial cells in angiogenesis dynamics

Abstract

Research into angiogenesis has contributed to progress in the fast-moving field of regenerative medicine. Designing coculture systems is deemed a helpful method to understand the dynamic interaction of various cells involved in the angiogenesis process. We investigated the juxtacrine and paracrine interaction between 3 different cells, namely rat marrow-derived mesenchymal stem cells (rMSCs), rat muscle-derived satellite cells (rSCs), and rat neonatal cardiomyocytes (rCMs), and endothelial cells (ECs) during angiogenesis process. In vitro Matrigel angiogenesis assay was performed whereby ECs were monocultured or cocultured with rMSCs, rSCs, and rCMs or their conditioned media (CM). In addition, in vivo Matrigel plug assay for angiogenesis was conducted to assess the angiogenic potential of the rCM-, rMSC-, and rSC-derived CM. Our results demonstrated that the rMSCs, rSCs, and rCMs elongated along the EC tubules, whereas the rMSCs formed tube-like structures with sprouting tip cells, leading to improved angiogenesis in the coculture system. Moreover, the rMSC- and rSC-derived CM significantly improved angiogenesis tube formation on Matrigel, accelerated EC chemotaxis, and increased the arteriolar density, vascularization index, and vascularization flow index in the Matrigel plug in vivo. Western blotting showed that rMSCs secreted a high level of vascular endothelial growth factor, basic fibroblast growth factor, and stromal-derived factor-1-alpha. Tie2 is also shed from rMSCs. This study demonstrated that stem cells interact with ECs in the juxtacrine and paracrine manner during angiogenesis, and marrow MSCs have superior angiogenic properties.

FIG. 1.

Orthogonal and reconstructed 3D ultrasonography imaging of Matrigel mass that was injected subcutaneously. The swollen form of Matrigel beneath the skin during injection changed and flattened by day 3 or 7 (upper right part in this figure). After ultrasonography imaging of the injection site, the hypoechoic mass was selected, and 3D power Doppler information was measured by Vocal analysis. In the bottom right, the 3D reconstructed image of the gel is shown. This image is related to Matrigel impregnated with control Dulbecco's modified Eagle's medium (DMEM) at day 7. Red points that are seen in the gel are indicative of blood vessels. On the left side, the upper column is indicative of gray scale g (0–100), and the lower column represents color value c (0–100). Color images available online at www.liebertpub.com/scd

FIG. 2.

Monoculture systems. Phase-contrast and fluorescent micrographs of rSCs^GFP+/+, rCMs^GFP+/+, and rMSCs^GFP+/+, which were separately cultured on Matrigel. As is seen, these cells formed cellular aggregates, without capability to form tube-like vascular structures alone. Scale bars: 100 μm. rSCs, rat muscle-derived satellite cells; rCMs, rat neonatal cardiomyocytes; rMSCs, rat marrow-derived mesenchymal stem cells. Color images available online at www.liebertpub.com/scd

FIG. 3.

Representatives of tube formation by ECs^CM-Dil+ cocultured with rCMs^GFP+/+, rSCs^GFP+/+, rMSCs^GFP+/+, or in combined cell culture (A–D). In all cocultures, GFP-positive cells align along the red-fluorescent-labeled ECs to promote vascular network formation. GFP-positive sprouting tip cells with a propensity for tube-like formation were seen prominently in the rMSC-EC coculture (C). In the combined cell culture (ECs–rMSCs–rSCs–rCMs), dual-labeled cells (yellow) are seen by dual-fluorescence filter, indicating cellular crosstalk (D). ECs, endothelial cells; GFP, green fluorescent protein. Color images available online at www.liebertpub.com/scd

FIG. 4.

Improvement in Matrigel angiogenesis in cocultures compared to EC monoculture. Coculture of rMSCs with ECs significantly increased the angiogenesis scoring (A), and Matrigel tube formation (B, C). Data are expressed as mean±standard error. *P<0.05, **P<0.005.

FIG. 5.

Angiogenesis scoring (A, colony scoring per 25 colonies) and tube formation (B, tube length [μm]); (C tube area [pixel²]) on Matrigel by ECs incubated with the cell CM. The rMSC-derived CM improved angiogenesis tube formation on Matrigel. *P<0.05, **P<0.005. CM, conditioned media.

FIG. 6.

Effect of cell-derived CM on EC migration. The rMSC-derived CM significantly enhanced EC migration. Data are mean±standard deviation of the number of migrated cells per high-power field (HPF).*P<0.05, **P<0.005.

FIG. 7.

Representatives of the 3D views of Matrigel plugs by Vocal^® analysis in the 4 groups at day 3 (left column) and day 7 (right column). [(A) Control DMEM; (B) rCM-derived CM; (C) rSC-derived CM; and (D) rMSC-derived CM]. Blood vessels started to develop from the periphery of the Matrigel plug with more vascular density in the ventral aspect of the plug. In the control group, the vascular density is less than that of the other groups. The highest vascular density is seen in the rMSC- and rSC-derived CM groups. Vessel growth into Matrigel was analyzed by ultrasonography imaging and presented as vascularization index (VI) (E) and vascularization flow index (VFI) (F). The rMSC- and rSC-derived CM substantially improved both the VI and VFI indices 7 days postinjection of Matrigel containing CM (*P<0.05, **P<0.005).

FIG. 8.

The effect of cell-derived CM on the development of α-SMA-positive arterioles, vWF-positive capillary ECs, and recruitment of myofibroblasts in the Matrigel plugs (A–K). The rMSC-derived CM enhanced arteriolar development in the Matrigel plug (D, I). Myofibroblasts recruitment (arrows) was most prominent in the rSC-derived CM group (C, J). Capillary density was markedly augmented in the Matrigels impregnated with the rSC- (G, K) as well as rMSC-derived CM (H, K). Mononuclear cell infiltration is noticeable in the Matrigels containing the rCM-derived CM (B, F). (A, E) Refer to control DMEM (*P<0.05, **P<0.005). α-SMA, alpha-smooth muscle actin; vWF, von Willebrand factor. Color images available online at www.liebertpub.com/scd

FIG. 9.

Western blot analysis for angiogenic paracrine factors in cell-derived CM. VEGF was found in the CM from the rMSCs, rSCs, and rCMs with different levels. The highest VEGF level was detected in the rMSC-derived CM and the lowest level in the rCM-derived CM (A). bFGF was detected in the rSC- and rMSC-derived CM (B). sTie2 was present only in the rMSC-derived CM with no shedding into the CM of rSCs and rCMs (C). The presence of SDF-1α was also detected only in the CM secreted by the rMSCs (D). DMEM was used as negative control. Data are from 3 independent experiments. VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; sTie2, soluble fragment of Tie2; SDF-1α, stromal-derived factor-1-alpha.