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. 2018 Jul 2;9:1517. doi: 10.3389/fimmu.2018.01517

Figure 4.

Figure 4

Isolation and characterization of CD31+ myofibrocytes from CD31-TFPI-Tg mice. (A) Immunocytofluorescence analysis of CD34+ cells from wild-type (WT) (black bars) or CD31-TFPI-Tg (white bars) mice compared to the purified fraction of CD34+ cells that express human tissue factor pathway inhibitor (hTFPI) on the cell surface (gray bars). Cells were stained with antibodies against anti-smooth muscle actin and either collagen-1 or CD31, and the proportion of cells expressing each was determined. Data are derived from counting at least 100 stained cells from each of five different animals. (B) Thrombin generation assay: CD34+ cells from WT (white bars) or purified CD31+ myofibrocytes from CD31-TFPI-Tg (black bars) were incubated with FXa, FVa, and prothrombin as described in Section “Materials and Methods,” and supernatants harvested at the times indicated for analysis of the amount of thrombin generated. (C–G) Comparison of the functional impact, after adoptive transfer, of the cell-surface hTFPI+ and hTFPI-negative fractions of CD34+ cells from CD31-TFPI-Tg mice. (C,D) Cross sectional images of WT carotid artery 28 days post-injury stained with elastin van Gieson’s stain after adoptive transfer of cell-surface hTFPI+ fraction (C) or hTFPI-negative fraction (D) on day of injury. (E) Neointimal area (left panel) and intima:media ratio (right panel) of vessels taken from WT animals 28 days post-injury after adoptive transfer of 1 × 106 hTFPI-negative (black bars) or hTFPI-positive (white bars) fractions. Data derived from examination of three random sections from five different vessels. *p < 0.01. (F,G) Immunohistology of sections through WT injured mouse carotid arteries harvested 28 days post-injury. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and (red) anti-collagen-1 and (green) anti-hTFPI. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. Mice received cell-surface hTFPI-negative fraction (F) or cell-surface hTFPI+ fraction (G) of CD34+ cells on day of injury. Table besides panels (F,G) describes summary of staining from all mice (n = 6), showing the proportion of the neointimal area that is positive for collagen-1, hTFPI, or both on day 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods). Subtracting the proportional area occupied by dual positive cells from the total area occupied by hTFPI+ cells gives the proportional area occupied by non-adoptively transferred collagen-1-positive cells. *cf hTFPI-negative fraction p = NS. All experiments repeated at least twice.