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. 2012;7(2):e32815.
doi: 10.1371/journal.pone.0032815. Epub 2012 Feb 28.

Bone marrow-derived progenitor cells augment venous remodeling in a mouse dorsal skinfold chamber model

Megan E Doyle  1 , Jeffrey P Perley, Thomas C Skalak
Affiliations

Bone marrow-derived progenitor cells augment venous remodeling in a mouse dorsal skinfold chamber model

Megan E Doyle et al. PLoS One. 2012.

Abstract

The delivery of bone marrow-derived cells (BMDCs) has been widely used to stimulate angiogenesis and arteriogenesis. We identified a progenitor-enriched subpopulation of BMDCs that is able to augment venular remodeling, a generally unexplored area in microvascular research. Two populations of BMDCs, whole bone marrow (WBM) and Lin(-)/Sca-1(+) progenitor cells, were encapsulated in sodium alginate and delivered to a mouse dorsal skinfold chamber model. Upon observation that encapsulated Sca-1(+) progenitor cells enhance venular remodeling, the cells and tissue were analyzed on structural and molecular levels. Venule walls were thickened and contained more nuclei after Sca-1(+) progenitor cell delivery. In addition, progenitors expressed mRNA transcript levels of chemokine (C-X-C motif) ligand 2 (CXCL2) and interferon gamma (IFNγ) that are over 5-fold higher compared to WBM. Tissues that received progenitors expressed significantly higher protein levels of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and platelet derived growth factor-BB (PDGF-BB) compared to tissues that received an alginate control construct. Nine days following cell delivery, tissue from progenitor recipients contained 39% more CD45(+) leukocytes, suggesting that these cells may enhance venular remodeling through the modulation of the local immune environment. Results show that different BMDC populations elicit different microvascular responses. In this model, Sca-1(+) progenitor cell-derived CXCL2 and IFNγ may mediate venule enlargement via modulation of the local inflammatory environment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Microvascular Remodeling in the Mouse Dorsal Window Chamber.
(A) Representative montage of the window chamber at 4x. (B) Example ten day time course of the remodeling network. Arterioles and venules enlarge slightly from days one to four. Diameter increases and angiogenic remodeling is accelerated from days 4 to 10.
Figure 2
Figure 2. Encapsulated Cells' Impact on Arteriole and Venule Diameters over 10 Days.
(A) Average absolute arteriole diameter change over ten days in vessels with starting diameter below the mean. (B) Average absolute arteriole diameter change over ten days in vessels with starting diameter above the mean. (C) Average absolute venule diameter change over ten days in vessels with starting diameter below the mean. (D) Average absolute venule diameter change over ten days in vessels with starting diameter above the mean. * indicates p<0.05 as calculated by ANOVA. Data are means +/− S.E.
Figure 3
Figure 3. Structural Analysis and Cellular Composition of Remodeled Venules.
(A) Average venule wall area per micron inner luminal circumference. (B) Number of nuclei in venule wall per micron inner luminal circumference. (C) Number of cell nuclei per square micron venule wall area. (D) Number of CD45 positive cells per micron luminal actin length. * indicates p<0.05 as calculated by Student's t-test. Data are means +/− S.E.
Figure 4
Figure 4. Statistical Analysis of mRNA Transcripts from Multiple Cell Isolations.
Fold differences between mRNA transcript levels were compared for two isolations of the WBM and progenitor populations. The black line indicates no fold-change in gene expression. Pink lines indicate a five-fold change in gene expression threshold. The blue line indicates a threshold of statistical significance indicated by p<0.05 as calculated by Student's t-test.
Figure 5
Figure 5. Comparison of Negative Threshold to Cycle Number of Gene Amplification for WBM and Sca-1+ Progenitors.
Fn-1, IFN γ, and CXCL2 amplify at cycles significantly below the negative threshold in both WBM and Sca-1+ progenitors, while FGF-2 amplifies at a cycle number indicative of low genetic expression in both cell types. * indicates p<0.05 as calculated by ANOVA. Data are means +/− S.E.
Figure 6
Figure 6. Enhanced Protein Expression of VEGF, MCP-1, and PDGF-BB in Dorsal Skinfold Chambers.
(A) Delivery of encapsulated Sca-1+ progenitor cells elevated VEGF protein expression in dorsal tissue at day 4. (B) Delivery of encapsulated Sca-1+ progenitor cells elevated MCP-1 protein expression in dorsal tissue at day 4. (C) Delivery of encapsulated Sca-1+ progenitor cells elevated PDGF-BB protein expression in dorsal tissue at day 4. * indicates p<0.05 as calculated by ANOVA. Data are means +/− S.E.
Figure 7
Figure 7. Inflammatory Cell Recruitment in Sca-1+ Progenitor Cell Recipients and Control Animals.
(A) Number of CD45 positive cells per square micron skinfold chamber tissue. (B) Total percentage of CD45 expressing cells in the skinfold chamber tissue. * indicates p<0.05 as calculated by ANOVA. Data are means +/− S.E.
Figure 8
Figure 8. MCP-1 Expression in CD45+ Cells in Sca-1+ Progenitor Cell Recipients.
(A–C) CD45 expression in a cell 5 microns above and below the focal plane. (D–F) MCP-1 expression in a cell 5 microns above and below the focal plane. (G–H) Merged CD45 and MCP-1 expression in a cell 5 microns above and below the focal plane. (Scale bar, 5 µm).
Figure 9
Figure 9. Proposed Mechanism of Venular Remodeling in Response to Sca-1+ Progenitor Cells.
Processes by which CXCL2 and IFNγ derived from delivered Sca-1+ progenitor cells may stimulate the remodeling of venules in the dorsal skinfold window chamber. Boxes highlight processes that have led to venular structural adaptation.
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