Origin of Matrix-Producing Cells That Contribute to Aortic Fibrosis in Hypertension

Abstract

Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1(+)) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1(+) progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1(+). Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I(+) cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1(+) progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix.

Keywords: adventitia; aorta; flow cytometer; hypertension; inflammation.

Figure 1

Subsets of aortic cells that express matrix protein mRNAs in hypertension. A) CD31⁺, CD31⁻Sca-1⁺ and CD31⁻Sca-1⁻ cells were isolated using magnetic-activated cell sorting. B–E) Real-time PCR analysis of expression of collagen 1a1, 3a1, 5a1 and fibronectin-1 in cells fractions isolated from the aorta of sham and angiotensin II-treated mice (n=6).

Figure 2

Flow cytometric analysis of aortic collagen producing cells. Mice received angiotensin II or sham treatment for 14 days and flow cytometry was performed on single cells suspensions of the aorta. Panel A shows the gating strategy employed. Panels B–E show subsets of vascular cells that stain positively for intracellular collagen. Mean data are shown in panels F–I. Comparisons were made with Student’s t test (n=6–8).

Figure 3

The role of Sca-1⁺ cells in adventitial collagen deposition. Tg^Ly-6A/EGFP mice received angiotensin II or sham treatment for 14 days. Frozen sections of mouse aortas were used for staining. A–C) Immunofluorescence showing co-localization of Sca-1 driven EGFP and collagen I in the aorta adventitia (n=6). D–G) Immunofluorescence showing co-localization of Sca-1 driven EGFP and collagen III and V in the aorta adventitia (n=6). Data in panels B, E and G represent percentages of DAPI⁺ cells.

Figure 4

The roles of cells derived from endothelial lineage or smooth muscle cell lineages. A–C) Lineage tracing studies using Tie1-cre × ROSA26-STOP^fl/fl-EYFP mice to identify cells of endothelial origin that produce collagen I (n=6). D–E) Lineage tracing using tg^myh11-cre × ROSA26-STOP^fl/fl-EYFP mice showing absence of collagen I producing cells of smooth muscle origin. Cre^−/− × ROSA26-STOP^fl/fl-EYFP littermates served as a negative control of EYFP expression.

Figure 5

Role of bone marrow derived cells in vascular fibrosis and summary. Mice underwent bone marrow transplant with EGFP⁺ cells and subsequently received sham or angiotensin II infusion. A–C) Co-localization of bone marrow derived cells with collagen and Fsp-1⁺ cells. D–I) Flow cytometric analysis of bone marrow derived cells in the aorta that produce collagen and express Sca-1. Gating strategy is shown in panel D.

Figure 6

Working hypothesis showing the contributions of Sca-1⁺ progenitors, bone marrow derived and cells of endothelial origin in aortic collagen deposition. The Venn diagram shows the relative contributions of various sources of collagen I-producing cells in the bone marrow chimeric mice.