High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization

Abstract

Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

Keywords: endothelial clusters; endothelial progenitor cells; high proliferative potential endothelial colony-forming cells; pulmonary hypertension; vascular remodeling.

Fig. 1.

Vasa vasorum (VV) neovascularization in pulmonary arteries (PA) of normal and pulmonary hypertensive animals. Hematoxylin and eosin (H&E) staining of lung sections of control (A) and hypoxic (B) calves demonstrates that thickening of the PA wall is associated with an apparent increase in the density of VV (green arrows) in vessels from hypoxic animals. Aw, airways; scale bar, 20 μm. In more advanced stages of the pulmonary hypertensive process in the “brisket disease” calf model of PH (see materials and methods), further expansion of VV [in adventitia (Adv), media (M), and even neointima (Int)] is observed (C). Demarcation lines identify the border between the neointimal and medial layers (as defined by the basal elastic lamellae). Scale bar, 100 μm. Immunofluorescence analysis of endothelial and progenitor cell marker expression in main pulmonary artery (MPA) VV shows the expression of CD31⁺ cells (D and G; ×10 magnification), CD31⁺CD133⁺ cells (E and H; ×40 magnification), and CD31⁺ CD34⁺ cells (F and I; ×40 magnification) in VV in sections from control (Co) and chronically hypoxic (Hx) animals.

Fig. 2.

Endothelial and progenitor cell marker expression in VV endothelial cells (VVEC). Quantitative RT-PCR analysis was performed on total cellular mRNA isolated from VVEC-Co and VVEC-Hx populations. Expression level of CD31, CD34, CD133, CD105, and c-kit was calculated relative to bovine 18S RNA in VVEC-Co and VVEC-Hx. Data represent mean relative mRNA expression in VVEC-Co and VVEC-Hx; P = 0.0303 (CD31); P = 0.5303 (CD34); P = 0.8763 (CD133); P = 0.0303 (CD105); P = 0.0408 (c-kit); *P < 0.05 (Student's t-test, followed by a Mann-Whitney posttest).

Fig. 3.

Quantitation of clonogenic and proliferative potential of single cell-derived colonies from VVEC, aortic endothelial cells (AOEC), and main pulmonary artery endothelial cells (MPAEC). Data represent the percentage of single cell-derived colonies from VVEC-Co (n = 3), VVEC-Hx (n = 3), AOEC-Hx (n = 3), and MPAEC-Hx (n = 3) on day 14 after single cell sorting. Colonies of each cell population were analyzed from 4–5 96-well plates (384–480 wells in total). HPP-CFC, high proliferative potential colony-forming cells; LPP-CFC, low proliferative potential colony-forming cells; EC, endothelial clusters. **P = 0.008 and *** P = 0.0001 (Student's t-test, followed by a Mann-Whitney posttest).

Fig. 4.

Characterization of high proliferative potential colony-forming cells (HPP-CFC) morphology and telomerase activity. A: the graph shows relative telomerase activity measured using a TRAPEZE RT telomerase kit. Shown are the data from individual cell populations isolated from control (control) and chronically hypoxic (hypoxic) animals. VVEC (n = 3), HPP-CFC-Co (n = 7), and HPP-CFC-Hx (n = 11). B: bright-field images represent VVEC and HPP-CFC cultures isolated from control and chronically hypoxic animals.

Fig. 5.

Quantitative RT-PCR analysis of endothelial and progenitor cell marker expression in HPP-CFC compared with VVEC. Quantitative RT-PCR analysis was performed on total cellular mRNA isolated from VVEC-Co and VVEC-Hx populations. Expression levels of CD31, CD34, CD105, and CD133 mRNA were calculated relative to bovine 18S RNA in cell populations of control and hypoxic animals. Data represent mean relative mRNA expression; CD31: *P < 0.05, VVEC-Co vs. VVEC-Hx. c-kit: *P < 0.05, VVEC-Co vs. VVEC-Hx; ***P < 0.001, VVEC-Co vs. HPP-CFC-Co; #P < 0.05, HPP-CFC-Co vs. HPP-CFC-Hx; and **P < 0.01, HPP-CFC-Co vs. HPP-CFC-Hx. CD105: **P < 0.01, HPP-CFC-Co vs. HPP-CFC-Hx (CD105) (1-way ANOVA, followed by a Bonferroni posttest).

Fig. 6.

Characterization of migratory properties of VVEC and HPP-CFC. Migration was carried out using Boyden chamber assay as described in materials and methods. Growth-arrested cells were seeded on top of the transwell permeable support, and ADP (500 μM) was added to the lower compartment to initiate migration. Migrated cells were fixed in methanol and stained with 0.2% crystal violet. Filters with migrated cells were photographed under ×20 magnifications in a phase-contrast microscope (Nikon) at 3 random fields and counted using ImageJ software. Graph represents the mean ± SE of migrated cells treated or untreated with ADP; ***P < 0.0001, control vs. ADP-stimulated cells.

Fig. 7.

Tube formation responses in VVEC and HPP-VVEC. A: tube formation was performed in angiogenesis slides (ibidi) as described in materials and methods. Growth-arrested VVEC and HPP-CFC (17,000/well) were seeded on polymerized Growth Factor Reduced Matrigel and incubated for 6 h with or without ADP (100 μM). B: evaluation of tube formation responses in VVEC and HPP-CFC were carried out using the Image-Pro Premier 9.0 software. VVEC-Co and VVEC-Hx (n = 3), HPP-CFC-Co (n = 3) and HPP-CFC-Hx (n = 6), each in triplicate. *P < 0.05 and ***P < 0.0001, control vs. hypoxic cells; #P < 0.05, ##P < 0.01, and ###P < 0.001, control vs. ADP-stimulated cells.