Existence, functional impairment, and lung repair potential of endothelial colony-forming cells in oxygen-induced arrested alveolar growth

Abstract

Background: Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth.

Methods and results: Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension.

Conclusions: Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage.

Keywords: angiogenesis inducing agents; hypertension, pulmonary; lung diseases; stem cells.

Figure 1

ECFCs exist in the developing human fetal lung and are perturbed by hyperoxia. A, Phase-contrast microscopy showing characteristic cobblestone-like colonies of CD31-positive cells obtained by beads isolation. B, These cells demonstrate Dil-acLDL uptake (red) within 4 hours of incubation and Ulex europaeus-lectin binding (green) within 1 hour of incubation after fixation. Counterstaining with Hoechst 33258 (blue) illustrates that all adherent cells are positive for LDL uptake and U europaeus (Ulex)-lectin binding. C, These cells form tube-like structures when suspended in Matrigel. D, Fluorescent-activated cell sorting. Isolated cells are positive for endothelial-specific cell surface antigens CD31, CD105 (endoglin), CD144 (VE-cadherin), CD146 (M-CAM), and negative for monocyte/ macrophage–specific CD14 and hematopoietic cell–specific CD45. Filled gray histograms represent antigen staining with negative isotype controls overlaid in white. All experiments were performed in triplicate. E, Single-cell clonogenic assay. Single cells are capable of giving rise to clusters (up to 50 cells) or colonies 50 to 500 cells (low proliferative potential, LPP) or more than 500 cells (high proliferative potential, HPP) in 96-well plates when plated at a seeding density of 1 cell per well. Results represent the mean±standard error of mean of 3 independent experiments. F, On replating, HPP ECFCs were able to form clusters or secondary colonies with LPP and HPP. G, Subcutaneous Matrigel Plug Assay. Human fetal lung ECFCs form blood vessels de novo when seeded in fibronectin-collagen plugs (10⁶ ECFCs per implant) and implanted subcutaneously into the flanks of NOD/SCID mice. Fourteen days postimplantation, the cellularized implants were excised, paraffin embedded, and stained with hematoxylin and eosin and anti-human CD31 (brown). Black arrows indicate red blood cell–perfused anti-human CD31+ vessels within the gel implant. H, Hyperoxia impairs network formation in vitro. Human fetal lung ECFCs exposed to 40% hyperoxia in vitro show a significant decrease in the number of intersects in comparison with RA-exposed ECFCs (n=5 for each group, *P<0.05). I, Comparative single-cell clonogenic assay. Human fetal lung ECFC exposed to 40% hyperoxia in vitro and plated in 96-well plates at a seeding density of 1 cell per well formed clusters and gave rise to colonies with LPP, but formed significantly fewer HPP than RA-exposed ECFCs (*P<0.05, 3 independent experiments). Dil-acLDL indicates Dil-acetylated low-density lipoprotein; ECFC, endothelial colony-forming cell; LDL, low-density lipoprotein; NOD/SCID, nonobese diabetic/ severe combined immunodeficiency; and RA, room air.

Figure 2

ECFCs exist in the developing rat lung. A, Phase-contrast microscopy showing characteristic cobblestone-like colonies of CD31-positive cells obtained by beads isolation. B, These cells demonstrate Dil-acLDL uptake (red) within 4 hours of incubation and Ulex europaeus-lectin binding (green) within 1 hour of incubation after fixation. Counterstaining with Hoechst 33258 (blue) illustrates that all adherent cells are positive for LDL uptake and U europaeus (Ulex)-lectin binding. C, These cells form tube-like structures when suspended in Matrigel. D, Fluorescent-activated cell sorting. Isolated endothelial cells are positive for endothelial-specific cell surface antigens CD31, vWF, and VEGFR2 and negative for monocyte/macrophage–specific CD14 and hematopoietic cell–specific CD45 and CD133. E, Single-cell clonogenic assay. Rat lung endothelial cells are capable of giving rise to clusters (up to 50 cells) or colonies 50 to 500 cells (low proliferative potential, LPP) or >500 cells (high proliferative potential, HPP) in 96-well plates when plated at a seeding density of 1 cell per well. Results represent the mean±standard error of mean of 3 independent experiments. On replating, HPP ECFCs were able to form clusters or secondary colonies with LPP and HPP. F, Subcutaneous Matrigel Plug Assay. Rat lung ECFCs form blood vessels de novo when seeded on fibronectin-collagen plugs (10⁶ ECFCs per implant) and implanted subcutaneously into the flanks of NOD/SCID mice. Fourteen days postimplantation, the cellularized implants were excised, paraffin embedded, and stained with hematoxylin and eosin and anti-human CD31 (brown). The arrow points to a rat endothelial cell; the rat endothelial cell lines a vessel filled with mouse red blood cells indicating that the rat endothelial cells have connected with the mouse circulation. The arrowhead points to a murine endothelial cell lining a mouse blood vessel. Dil-acLDL indicates Dil-acetylated low-density lipoprotein; ECFC, endothelial colony-forming cell; LDL, low-density lipoprotein; and NOD/SCID, nonobese diabetic/severe combined immunodeficiency.

Figure 3

Rat lung ECFC function is perturbed in hyperoxia-induced experimental BPD in newborn rats. A, Schematic describing the rat from postnatal day (P) 4 to P14 and studied in comparison with model of hyperoxia-induced BPD. Newborn rats are housed in 95% O₂ room air (RA)–raised control rats. Exposure of rat pups to hyperoxia during the alveolar stage of lung development results in arrested alveolar growth characterized by larger and fewer alveolar structures as shown in hematoxylin and eosin–stained representative lung slides. ECFCs were isolated from RA and hyperoxia-exposed rat lungs on P14, and their proliferative, clonogenic, and vessel-forming potentials were assessed. B, MTT Assay. Lung ECFCs form RA and hyperoxic animals were plated at equal cell densities and cultured under identical culture conditions. ECFCs from the hyperoxia-exposed group showed decreased cell growth as assessed by MTT assay (n=6, P<0.05 on days 12 and 16, P<0.01 on day 8, and P<0.001 on day 20). C, Matrigel assay. Quantitative assessment of the ability to form tube-like structures on Matrigel reveals a significant decrease in the total cord length and the number of intersects in the hyperoxia-exposed ECFCs in comparison with RA ECFCs (n=6 for each group, *P<0.05). D, Comparative single-cell assay. Colony-forming potential of single-cell–plated rat lung ECFCs was assessed by measuring the percentage of single ECFCs capable of generating colonies after 14 days in culture. Significantly fewer ECFCs from the hyperoxia group are capable of generating colonies with ≥500 cells in comparison with RA controls (n=6 lungs/group, *P<0.05). BPD indicates bronchopulmonary dysplasia; ECFC, endothelial colony-forming cell; and MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 4

Human umbilical cord blood ECFC therapy improves lung function and reverses alveolar growth arrest in hyperoxia-exposed newborn mice. A, Experimental design. Newborn rag^−/− mice were housed in room air (RA) or 85% O₂ from postnatal day (P) 4 to P14. At P14, mice were treated with intrajugular injections of human umbilical cord blood–derived ECFCs and housed in RA until the assessment of end points on P28. B, Lung function testing. Untreated mice exposed to hyperoxia had significantly increased lung compliance in comparison with RA and ECFC-treated RA controls (n=4–7, P<0.05). Lung compliance was significantly improved in hyperoxia-exposed mice treated with ECFCs in comparison with untreated hyperoxia-exposed controls (P<0.05). C, Representative hematoxylin and eosin–stained lung sections showing larger and fewer alveoli in untreated hyperoxia-exposed (O₂) lungs in comparison with control mice housed in RA at P28. Intrajugular administration of ECFCs in O₂-exposed animals preserved alveolar growth. ECFCs did not affect lung structure in control RA mice. D, Quantitative assessment of alveolar architecture by mean linear intercept confirms the protective effect of ECFCs on alveolar growth (n=5/group, *P<0.05). BPD indicates bronchopulmonary dysplasia; ECFC, endothelial colony-forming cell; and hUCB, human umbilical cord blood.

Figure 5

Human umbilical cord ECFCs improve lung angiogenesis and prevent pulmonary hypertension in hyperoxia-exposed newborn mice. A and B, Effects of ECFC treatment on pulmonary vessel density assessed on lung slides stained with von Willebrand Factor at P28. The pulmonary vessel density of 30 to 100μm sized blood vessels per 10 high-power fields (×40) was significantly decreased in the lungs of O₂-exposed animals in comparison with RA. Intrajugular injection of ECFCs significantly improved pulmonary vessel density (n=5/group, *P<0.01). C, Pulmonary arterial acceleration time (PAAT) was significantly decreased in O₂-exposed animals in comparison with controls. Intrajugular ECFCs restored the PAAT almost to control levels (n=4–6/group, *P<0.01). D, Hyperoxia-exposed mice had significant RVH as indicated by the increase in right ventricle/left ventricle plus septum (RV/LV+S) ratio in comparison with controls. ECFC therapy significantly reduced RVH (n=4–9/group, *P<0.01). ECFC indicates endothelial colony-forming cell; HPF, high-power field; RA, room air; and RVH, right ventricle hypertrophy.

Figure 6

Low engraftment rate after intrajugular injection of human umbilical cord ECFCs. A, Quantitative RT-PCR for human Alu sequences in the lung revealed a rapid decline during the first day after injection. Human DNA became almost undetectable 3 days after injection. B, Representative frozen lung section at P21 depicting rare fluorescent-labeled ECFCs. ECFC indicates endothelial colony-forming cell; and RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 7

Paracrine effect of human umbilical cord ECFCs. A, Representative confluent monolayers of freshly isolated rat alveolar type 2 epithelial cells (AT2) damaged by the use of a pipette tip, washed to remove damaged cells, and treated with DMEM, ECFC-derived conditioned medium (CdM) or HUVEC CdM. B, The percentage of wound closure showed that ECFC CdM accelerated AT2 wound closure in comparison with DMEM and HUVEC CdM (n=6/group, *P<0.05). C, Representative endothelial network formation assay on Matrigel of human fetal lung ECFCs treated with DMEM, ECFC-derived CdM or HUVEC CdM in room air (RA) and hyperoxia (O₂). D, Quantitative assessment of cordlike structure formation shows a significant decrease in the number of intersects in hyperoxia in DMEM and HUVEC CdM–treated ECFCs in comparison with RA-exposed ECFC. ECFC CdM preserved the number of intersects in hyperoxia (n=3–6/group, *P<0.05). E, Representative hematoxylin and eosin section showing arrested alveolar growth in O₂-exposed newborn rats in comparison with RA controls. ECFC CdM significantly preserved alveolar growth. F, Quantitative assessment of the mean linear intercept confirms the protective effect of ECFC CdM on alveolar growth (n=6/group, *P<0.001). G, Mean data showing decreased pulmonary vessel density in O₂-exposed newborn rats in comparison with RA controls as assessed by the number of barium-filled pulmonary vessels. ECFC CdM significantly attenuated the loss of pulmonary vessels in hyperoxia (n=5/group, *P<0.01). H, Pulmonary arterial acceleration time (PAAT) was significantly decreased in O₂-exposed animals in comparison with controls. ECFC CdM preserved the PAAT in comparison with untreated O₂-exposed animals (n=5–6/group, *P<0.05). I, Hyperoxia-exposed rats had significant RVH as indicated by the increase in right ventricle/left ventricle plus septum (RV/LV+S) ratio in comparison with controls. ECFC CdM significantly reduced RVH in comparison with untreated O₂-exposed animals (n=5–6/group, *P<0.05). DMEM indicates Dulbecco’s modified Eagle medium; ECFC, endothelial colony-forming cell; HUVEC, human umbilical endothelial cell; and RVH, right ventricle hypertrophy.

Figure 7

Paracrine effect of human umbilical cord ECFCs. A, Representative confluent monolayers of freshly isolated rat alveolar type 2 epithelial cells (AT2) damaged by the use of a pipette tip, washed to remove damaged cells, and treated with DMEM, ECFC-derived conditioned medium (CdM) or HUVEC CdM. B, The percentage of wound closure showed that ECFC CdM accelerated AT2 wound closure in comparison with DMEM and HUVEC CdM (n=6/group, *P<0.05). C, Representative endothelial network formation assay on Matrigel of human fetal lung ECFCs treated with DMEM, ECFC-derived CdM or HUVEC CdM in room air (RA) and hyperoxia (O₂). D, Quantitative assessment of cordlike structure formation shows a significant decrease in the number of intersects in hyperoxia in DMEM and HUVEC CdM–treated ECFCs in comparison with RA-exposed ECFC. ECFC CdM preserved the number of intersects in hyperoxia (n=3–6/group, *P<0.05). E, Representative hematoxylin and eosin section showing arrested alveolar growth in O₂-exposed newborn rats in comparison with RA controls. ECFC CdM significantly preserved alveolar growth. F, Quantitative assessment of the mean linear intercept confirms the protective effect of ECFC CdM on alveolar growth (n=6/group, *P<0.001). G, Mean data showing decreased pulmonary vessel density in O₂-exposed newborn rats in comparison with RA controls as assessed by the number of barium-filled pulmonary vessels. ECFC CdM significantly attenuated the loss of pulmonary vessels in hyperoxia (n=5/group, *P<0.01). H, Pulmonary arterial acceleration time (PAAT) was significantly decreased in O₂-exposed animals in comparison with controls. ECFC CdM preserved the PAAT in comparison with untreated O₂-exposed animals (n=5–6/group, *P<0.05). I, Hyperoxia-exposed rats had significant RVH as indicated by the increase in right ventricle/left ventricle plus septum (RV/LV+S) ratio in comparison with controls. ECFC CdM significantly reduced RVH in comparison with untreated O₂-exposed animals (n=5–6/group, *P<0.05). DMEM indicates Dulbecco’s modified Eagle medium; ECFC, endothelial colony-forming cell; HUVEC, human umbilical endothelial cell; and RVH, right ventricle hypertrophy.

Figure 8

Long-term safety and efficacy of ECFC cell therapy. A, Representative hematoxylin and eosin–stained lung sections at 10 months of age shows persistent alveolar simplification in hyperoxia-exposed animals in comparison with lungs from rats housed in room air (RA). Oxygen-exposed animals treated with ECFCs have improved lung histology. B, The mean linear intercept confirms arrested alveolar growth in untreated O₂-exposed animals in comparison with RA and RA+ECFC–treated animals and preserved alveolar structure with ECFC therapy in comparison with untreated O₂-exposed animals (n=3–6/group, *P<0.05). C, Oxygen-exposed animals experienced reduced exercise capacity in comparison with RA-housed animals. Oxygen-exposed animals treated with ECFC had improved exercise capacity (n=3–6 animals/group, *P<0.05). D, Oxygen-exposed animals had decreased pulmonary arterial acceleration time (PAAT) in comparison with RA and RA+ECFC animals. Oxygen-exposed animals treated with ECFC had improved PAAT in comparison with untreated O₂-exposed animals (n=3–6 animals/group, *P<0.05). E, Representative lung section of an ECFC-treated mouse lung at 10 months of age showing no presence of CD31-positive structures. In contrast, CD31 staining is abundant in a human adult lung. ECFC indicates endothelial colony-forming cell.