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Basic Sciences

Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function After Inflammatory Lung InjurySignificance

Sun-Zhong Mao, Xiaobing Ye, Gang Liu, Dongmei Song, Shu Fang Liu
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https://doi.org/10.1161/ATVBAHA.115.305519
Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:1635-1644
Originally published May 14, 2015
Sun-Zhong Mao
From the Centers for Heart and Lung Research and Pulmonary and Critical Care Medicine, the Feinstein Institute for Medical Research, Manhasset, NY (S.-Z.M., X.Y., G.L., D.S., S.F.L.); and Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, China (S.-Z.M., S.F.L.).
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Xiaobing Ye
From the Centers for Heart and Lung Research and Pulmonary and Critical Care Medicine, the Feinstein Institute for Medical Research, Manhasset, NY (S.-Z.M., X.Y., G.L., D.S., S.F.L.); and Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, China (S.-Z.M., S.F.L.).
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Gang Liu
From the Centers for Heart and Lung Research and Pulmonary and Critical Care Medicine, the Feinstein Institute for Medical Research, Manhasset, NY (S.-Z.M., X.Y., G.L., D.S., S.F.L.); and Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, China (S.-Z.M., S.F.L.).
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Dongmei Song
From the Centers for Heart and Lung Research and Pulmonary and Critical Care Medicine, the Feinstein Institute for Medical Research, Manhasset, NY (S.-Z.M., X.Y., G.L., D.S., S.F.L.); and Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, China (S.-Z.M., S.F.L.).
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Shu Fang Liu
From the Centers for Heart and Lung Research and Pulmonary and Critical Care Medicine, the Feinstein Institute for Medical Research, Manhasset, NY (S.-Z.M., X.Y., G.L., D.S., S.F.L.); and Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, China (S.-Z.M., S.F.L.).
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Abstract

Objective—Disruption of endothelial barrier integrity is a characteristic of many inflammatory conditions. However, the origin and function of endothelial cells (ECs) restoring endothelial barrier function remain unknown. This study defined the roles of resident ECs (RECs) and bone marrow–derived endothelial progenitor cells (BMDEPCs) in endothelial barrier restoration after endotoxemic lung injury.

Approach and Results—We generated mice that enable to quantify proliferating RECs or BMDEPCs and also to study the causal link between REC or BMDEPC proliferation and endothelial barrier restoration. Using these mouse models, we showed that endothelial barrier restoration was associated with increased REC and BMDEPC proliferation. RECs and BMDEPCs participate in barrier repair. Immunofluorescence staining demonstrated that RECs proliferate in situ on endothelial layer and that BMDEPCs are engrafted into endothelial layer of lung microvessels at the active barrier repair phase. In lungs, 8 weeks after lipopolysaccharide-induced injury, the number of REC-derived ECs (CD45−/CD31+/BrdU+/rtTA+) or BMDEPC-derived ECs (CD45−/CD31+/eNOS+/GFP+) increased by 22- or 121-fold, respectively. The suppression of REC or BMDEPC proliferation by blocking REC or BMDEPC intrinsic nuclear factor-κB at the barrier repair phase was associated with an augmented endothelial permeability and impeded endothelial barrier recovery. RECs and BMDEPCs contributed differently to endothelial barrier repair. In lungs, 8 weeks after lipopolysaccharide-induced injury, REC-derived ECs constituted 22%, but BMDEPC-derived ECs constituted only 3.7% of the total new ECs.

Conclusions—REC is a major and BMDEPC is a complementary source of new ECs in endothelial barrier restoration. RECs and BMDEPCs play important roles in endothelial barrier restoration after inflammatory lung injury.

  • endothelial cells
  • NF-κB

Disruption of endothelial barrier and increase in endothelial permeability are major features of acute lung injury (ALI) associated with sepsis, trauma, and hemorrhage.1–3 However, the mechanisms regulating endothelial barrier restoration are poorly understood. Previous studies showed that fox head box M1-regulated proliferation genes play important roles.4,5 However, the effector cells that mediated the repair function of fox head box M1 have not been identified. The origin and function of endothelial cells (ECs) in endothelial barrier restoration after inflammatory organ injury remain unknown.

Resident ECs (RECs) are long believed to be a major source of ECs in endothelial repair. Indeed, neighboring RECs were observed to sprout into denuded area after mechanical arterial endothelial denudation.6 However, the role of RECs in the restoration of endothelial barrier function after organ injury has not been studied. A causal link between REC proliferation and endothelial barrier restoration remains to be established. Bone morrow–derived endothelial progenitor cells (BMDEPCs) as a source of ECs in angiogenesis and endothelial repair have been extensively studied, although whether BMDEPCs contribute to endothelial or vascular wall is controversial.7–16 Depending on type, nature, and severity of the injury, BMDEPCs were reported to contribute to endothelial and vascular repair in some experimental models7–13 but play no role in other models.13–16 Microvascular injury associated with septic ALI differs significantly from models used in previous reports in nature and severity. The contribution of BMDEPCs to endothelial barrier repair in septic organ injury is still unclear. Increased EPC mobilization and recruitment were observed in patients17,18 and animal models8 with septic organ injury. Autologous transplantation of EPCs suppressed lung inflammation, attenuated endothelial permeability, and lung edema and improved outcomes.19,20 However, it remains unclear whether the exogenous EPCs improve endothelial barrier function by preventing endothelial injury or by promoting endothelial repair, or by both. Other investigators have demonstrated that exogenously administered EPCs or other stem/progenitor cells did not participate in endothelial repair but alleviated organ injury and improved outcomes by immunomodulating and paracrine mechanisms.21–24 Furthermore, no previous study has examined the causal link between BMDEPC recruitment/proliferation and endothelial barrier restoration. Thus, whether BMDEPC is a source of ECs in endothelial barrier restoration remains unclear.

To study the causal link between REC or BMDEPC proliferation and endothelial barrier restoration, we need to examine the functional effect of inhibiting REC or BMDEPC proliferation on endothelial barrier restoration. In addition, barrier injury and repair are inter-related. Severity of injury determines the extent of repair. It is ideal to inhibit REC or BMDEPC proliferation only at the barrier repair phase. Animal model that enables to selectively inhibit REC or BMDEPC proliferation at the barrier repair phase are needed. No such an animal model has been reported.

In this study, we took advantage of the fact that endothelial repair depends mainly on proliferation of EC precursor cells and that the nuclear factor-κB (NF-κB) pathway is a major pathway controlling cell proliferation.25–30 We created EC-I-κBα-WT-BM or WT-EC-I-κBα-BM chimeric mice with doxycycline-inducible and REC- or BMDEPC-restricted overexpression of a mutant I-κBα (I-κBαmt). By treating these mice with doxycycline after peak of lung injury, we were able to inhibit REC or BMDEPC proliferation at the barrier repair phase by blocking REC or BMDEPC intrinsic NF-κB activity in a cell-targeted and stage-specific manner. By performing cause-to-effect studies in combination with cell fate mapping, we demonstrated that REC is a major and BMDEPC is a complementary source of new ECs in endothelial barrier repair and that RECs and BMDEPCs play important roles in endothelial barrier restoration after inflammatory ALI.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Active Endothelial Barrier Repair Occurs at 48 Hours

Endothelial permeability increased progressively between 0 and 24 hours, associated with increasing numbers of apoptotic ECs, and decreased progressively between 24 and 96 hours, associated with decreasing numbers of apoptotic ECs and increasing numbers of proliferating ECs (Figure 1A).30 On the basis of these observations, we defined 0 to 24 and 24 to 96 hours, respectively, as endothelial barrier injury and repair phases. At 48 hours, endothelial permeability decreased rapidly, associated with the highest level of EC proliferation (Figure 1A).30 We considered 48 hours as the active barrier repair phase and focused our subsequent studies on 48 hours.

Figure 1.
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Figure 1.

Endothelial barrier recovery is associated with increased resident endothelial cell (REC) and bone marrow–derived endothelial progenitor (BMDEPC) proliferation. A, Endothelial permeability in lungs was measured using Evan blue dye (EBD) leakage index at indicated time (hours) after saline (Con) or lipopolysaccharide (LPS; 5 mg/kg IP) injection. Mean±SEM of 6 mice per group. *P<0.05, compared with control. B–D, Lung cryosections were prepared at indicated time (hours) after LPS and 4 hours after 5-bromo-2-deoxyuridine (BrdU) injection, stained with BrdU+reverse tetracycline transactivator (rtTA), BrdU+green fluorescence protein (GFP) or GFP antibodies, and nuclei counterstained with 4,6-diamidino-2-phenylindole (DAPI). The number of BrdU+/rtTA+ proliferating RECs (B), BrdU+/GFP+ proliferating BMDEPCs (C), or GFP+ recruited BMDEPCs (D) was counted and expressed as a percentage of total cells as revealed by DAPI nuclear staining. Mean±SEM of 5 mice per group. *P<0.05, compared with controls.

Endothelial Barrier Recovery Is Associated With Increased REC and BMDEPC Proliferation

If RECs and BMDEPCs play important roles in endothelial barrier repair, the number of proliferating RECs or BMDEPCs should increase at the active repair phase. To track proliferating RECs or BMDEPCs in vivo, we generated EC-rtTA-GFP-BM chimeras by transplanting lethally irradiated EC-rtTA mice31 with bone marrows (BMs) from Tie2-GFP mice (Tables I and II in the online-only Data Supplement). These chimeras overexpress the reverse tetracycline transactivator (rtTA) on RECs and green fluorescent protein (GFP) on BMDEPCs. Fluorescence-activated cell sorting (FACS) analysis of BM mononuclear cells from donors and chimeras 2 months later confirmed that 95% of BM cells in the chimeras were donor BM origin (Figure IA in the online-only Data Supplement). FACS analysis of peripheral blood mononuclear cells further confirmed the high degree of BM chimerism in the EC-rtTA-GFP-BM mice (data not shown).

The endothelial-specific Tie2 promoter drives GFP expression in endothelial lineage cells. However, Tie2 was reported to be expressed on a subset of monocytes/macrophages.32 To clarify the percentage of monocytes in the GFP+ cell population in the lungs, we phenotyped GFP+ cells from lungs of Tie2-GFP donor mice 48 hours after lipopolysaccharide challenge. We found that 99% of the GFP+ cells are CD45−/CD31+/eNOS+ endothelial lineage cells, and <1% of them are CD45+/CD31+ hematopoietic cells (Figure II in the online-only Data Supplement). Our finding is consistent with a previous report.15 Thus, GFP+ cells in the EC-rtTA-GFP-BM mice represent principally BMDEPCs.

Two months after BM transplantation, mice were injected with saline or lipopolysaccharide and then with bromodeoxyuridine (BrdU) to label proliferating cells in vivo. Proliferating RECs or BMDEPCs, or recruited BMDEPCs, were identified by BrdU/rtTA or BrdU/GFP double staining, or by GFP staining, and counted. Time-course analyses showed that endothelial barrier recovery was associated with remarkably increased REC and BMDEPC proliferation (Figure 1). In particular, the high numbers of proliferating RECs and BMDEPCs at 48 hours were concordant with 48 hours being active barrier repair phase. No significant numbers of proliferating RECs and BMDEPCs were detected at 12 (injury phase) or 96 hours (barrier function recovered; Figure 1). ALI was associated with an increased recruitment of GFP+ BMDEPCs in lungs (Figure 1D). However, BMDEPC recruitment was not correlated with barrier recovery but correlated with barrier injury (Figure 1A versus 1D), suggesting that ALI stimulates BMDEPC recruitment.

RECs Participate in Endothelial Barrier Repair

If RECs participate in endothelial barrier repair, these cells should proliferate in situ on endothelial layer at the active repair phase to give rise to new ECs. Furthermore, the REC-derived daughter ECs should significantly increase in lungs after recovery from injury. EC-rtTA-GFP-BM mice that overexpress rtTA only on RECs (Table II in the online-only Data Supplement) were injected with BrdU at 44 hours after lipopolysaccharide injection to label proliferating cells. Lungs were harvested at 48 hours or at 8 weeks after lipopolysaccharide injection to track the location of proliferating RECs or to quantify the REC-derived new ECs in lungs. We visualized endothelial layer by immunofluorescence staining of lung sections with rtTA or CD31 antibody. We identified proliferating RECs by BrdU and rtTA double immunofluorescence staining. Confocal microscopic examination revealed that BrdU+/rtTA+ proliferating RECs were localized on the endothelial layer of microvessels (Figure 2A). The BrdU+/rtTA+ proliferating RECs coexpressed EC marker, CD31, and were localized on the CD31+ endothelial layer but were not localized on the aquaporin-5 (Aqu5)+ epithelial layer (Figure 2A). This result provides histological evidence that RECs proliferate in situ on endothelial layer at the active barrier repair phase.

Figure 2.
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Figure 2.

Resident endothelial cells (RECs) participate in endothelial repair. A, RECs proliferate in situ on the endothelial layer at the active repair phase. Lung sections from mice 48 hours after lipopolysaccharide (LPS) injection were stained with antibodies against proliferative marker, 5-bromo-2-deoxyuridine (BrdU), REC marker, reverse tetracycline transactivator (rtTA), EC marker, CD31, and alveolar epithelial cell marker, aquaporin-5 (Aqu5), and nuclei counterstained with TO-PRO-3 dye (Pro-3). 3D projections (A1–A6) or single images (A7–A10) of confocal z-stacks are shown. A1, BrdU+ staining (green) detects proliferating cells (light blue nuclei); blue, Pro-3 nuclear staining. A2, rtTA+ staining (red) detects RECs and visualizes the endothelial layer. A3, Merge of A1 and A2 shows BrdU+/rtTA+ RECs (arrow indicated) localized on rtTA+ endothelial layer of alveolar microvessels. A4 and A5, Orthogonal view (X–Y, X–Z, and Y–Z) of the boxed area in A3 at higher magnification confirms colocalization of BrdU+ and rtTA+ signals and colocalization of BrdU+ and Pro-3+ stainings. Note, the blue nuclear staining in A4 or the red rtTA staining in A5 was omitted for clarity. A6 and A7, BrdU+/CD31+ RECs (arrow indicated) are localized on CD31+ endothelial layer of alveolar microvessels. A8–A10, Higher magnification of the boxed area in A7 is shown. A8, BrdU (green) and CD31 (red) double stain shows that BrdU+ proliferating REC is localized on CD31+ endothelial layer (red). A9, BrdU (green) and Aqu5 (blue) double stain shows that BrdU+ proliferating REC is not localized on Aqu5+ epithelial layer (blue). A10, Merge of A8 and A9 confirms that BrdU+ REC is localized on the endothelial layer (red) between 2 epithelial layers (blue). Scale bars, 40 µm (A1, A2, A3, A6 and A7), 8 µm (A4 and A5), and 3 µm (A8, A9, and A10). Fluorescence-activated cell sorting pictures (B) and bar graph (C) show an increased number of REC-derived ECs, defined as CD45−/CD31+/rtTA+/BrdU+ cells, in lungs of mice 8 weeks after LPS injection, compared with saline-injected mice (Con). Mean±SEM of 5 mice per group. *P<0.05, compared with control.

FACS analysis showed that the number of REC-derived new ECs (CD45−/CD31+/BrdU+/rtTA+) was ≈22-fold higher in lungs of EC-rtTA-GFP-BM mice 8 weeks after lipopolysaccharide-induced injury, compared with lungs from mice 8 weeks after saline injection (Figure 2B and 2C). These results provide cytological evidence for REC’s participation in endothelial barrier repair.

BMDEPCs Contribute to Endothelial Barrier Repair

BMDEPC incorporation into endothelial layer is a critical step in BMDEPC-mediated endothelial repair. To seek histological evidence of BMDEPC engraftment, we stained lung sections from mice 48 hours after lipopolysaccharide injection with antibodies against BMDEPC marker, GFP, EC markers, CD31 and Ve-cadherin (Ve), or alveolar epithelial cell marker, Aqu5. Confocal microscopic examination identified GFP+/CD31+ BMDEPCs localized on the CD31+ endothelial layer of lung microvessels (Figure 3A). The GFP+ BMDEPCs were also Ve+ and localized on the Ve+ endothelial layer but not localized on the Aqu5+ alveolar epithelial layer (Figure 3A). This result provides evidence for BMDEPC engraftment into the endothelial layer in the lung at the active repair phase.

Figure 3.
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Figure 3.

Bone marrow–derived endothelial progenitor cells (BMDEPCs) contribute to endothelial repair. A, BMDEPCs engraft into microvessel wall. Lung sections from mice 48 hours after lipopolysaccharide (LPS) injection were stained with antibodies against BMDEPC marker, green fluorescent protein (GFP), endothelial cell (EC) markers, CD31 and Ve-cadherin (Ve), alveolar epithelial cell marker (Aqu5), and nuclei counterstained with Pro-3 dye. 3D projections (A1 to A6) or single images (A7 to A10) of confocal z-stacks are shown. A1, GFP+ (green) staining identifies BMDEPCs. A2, CD31+ (red) staining visualizes the endothelial layer. A3, Merge of A1 and A2 shows engrafted (GFP+/CD31+) BMDEPCs (yellow cells) localized on CD31+ endothelial layer (red). A4 and A5, 3D (A4) or orthogonal (A5) projection of the boxed area in A3 at higher magnification confirms GFP/CD31 coexpression on the same individual BMDEPC. For clarity, the blue nuclear staining was omitted. A6 and A7, GFP+/Ve+ BMDEPCs (arrow indicated) are localized on Ve+ endothelial (red) but not on Aqu5+ epithelial (blue) layer. High magnification of the boxed area in A7 is shown in A8–A10. A8, GFP (green) and Ve (red) double staining shows that GFP+ BMDEPCs express Ve (yellow cells) and are localized on Ve+ endothelial layer. A9, GFP (green) and Aqu5 (red) double staining shows that GFP+ BMDEPCs do not express Aqu5 (green cells) and are not localized on Aqu5+ epithelial layer. A10, GFP (green), Ve (red), and Aqu5 (blue) triple staining confirms that GFP+ BMDEPCs (yellow cells) are localized on endothelial (red) but not on epithelial (blue) layer. Scale bars, 40 µm (A1, A2, A3, A6, and A7) and 8 µm (A4, A5, A8, A9, and A10). B and C, Fluorescence-activated cell sorting pictures (B) and bar graph (C) show increased numbers of GFP+/CD31+ cells in the CD45−/CD31+/eNOS+ EC population in lungs of mice 8 weeks after LPS injection, compared with lungs of mice 8 weeks after saline injection (Con), indicating an increased BMDEPC engraftment. Mean±SEM of 5 mice per group. *P<0.05, compared with control mice. eNOS indicates endothelial nitric oxide synthase.

We quantified the engrafted BMDEPCs (CD45-/CD31+/eNOS+/GFP+) in lungs from mice 8 weeks after saline or lipopolysaccharide injection, a time point when organ inflammation and inflammation-associated BMDEPC recruitment has been subsided. FACS analysis showed that the number of CD45−/CD31+/eNOS+/GFP+ cells was ≈121-fold higher in lipopolysaccharide-injected than in saline-injected lungs (Figure 3B and 3C), indicating an increased BMDEPC engraftment in injured lungs. These results provide cytological evidence for BMDEPC engraftment into lung microvessels and suggest that BMDEPC contribute to endothelial barrier repair.

Different Contributions of RECs Versus BMDEPCs to Endothelial Barrier Repair

We next compare the relative contributions of RECs versus BMDEPCs to endothelial barrier repair. We counted the numbers of proliferating RECs and BMDEPCs in lungs at 48 hours and the numbers of REC- and BMDEPC-derived new ECs in lungs 8 weeks after lipopolysaccharide-induced injury. At 48 hours, the number of proliferating RECs was 2.4-fold higher than proliferating BMDEPCs in lipopolysaccharide-challenged lungs, although both proliferating RECs and BMDEPCs were remarkably higher than control lungs (Figure 4A and 4B). In lungs, 8 weeks after lipopolysaccharide-induced injury, total number of new ECs, defined as CD45−/CD31+/BrdU+ cells, accounted for ≈2% of total lung cells, which is in agreement with our previous observation that apoptotic ECs accounted for ≈2% of total lung cells in this mouse model of ALI.30 Among the new EC subpopulation, 83% were rtTA+ (REC-derived), but only 9.5% were GFP+ (BMDEPC-derived; Figure 4C and 4D).

Figure 4.
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Figure 4.

Resident endothelial cells (RECs) and bone marrow–derived endothelial progenitor cells (BMDEPCs) contribute differently to endothelial barrier repair. A, Representative micrographs of reverse tetracycline transactivator (rtTA)+5-bromo-2-deoxyuridine (BrdU; RECs) or green fluorescence protein (GFP)+BrdU (BMDEPCs) double staining of lung sections from EC-rtTA-GFP-BM mice 48 hours after saline (Con) or lipopolysaccharide (LPS) injection. The number of rtTA+/BrdU+ proliferating RECs was significantly higher than the number of GFP+/BrdU+ proliferating BMDEPCs (arrows indicated). Scale bar, 75 µm. B, Bar graph shows higher number of proliferating RECs (red bars) than proliferating BMDEPCs (black bars) in lungs at 48 hours post LPS. Mean±SEM of 5 mice per group. *P<0.05, compared with BMDEPCs at 48 hours. C and D, Fluorescence-activated cell sorting pictures (C) and bar graph (D) show that lung cells from EC-rtTA-GFP-BM mice 8 weeks after LPS injection have a remarkably higher number of REC-derived (rtTA+/CD31+) than BMDEPC-derived (GFP+/CD31+) ECs in the CD45−/CD31+/BrdU+ new EC population. Mean±SEM of 5 mice per group. *P<0.05, compared with BMDEPCs. E, Bar graph shows higher number of REC-derived new ECs (CD45−/CD31+/BrdU+/rtTA+) than engrafted BMDEPCs (CD45−/CD31+/eNOS+/GFP+) in lung cells from EC-rtTA-GFP-BM mice 8 weeks after LPS injection. Mean±SEM of 5 mice per group. *P<0.05, compared with engrafted BMDEPCs.

BMDEPCs may replace apoptotic/dead ECs by differentiation.9 We compared the number of REC-derived ECs (CD45−/CD31+/BrdU+/rtTA+ cells) to the number of total engrafted BMDEPCs (CD45−/CD31+/eNOS+/GFP+ cells), which includes BrdU+ and BrdU−, BMDEPC-derived ECs, in lungs of EC-rtTA-GFP-BM mice (Table II in the online-only Data Supplement) 8 weeks after lipopolysaccharide injection. REC-derived ECs constituted 22%, but the total engrafted BMDEPCs constituted only 3.7% of the total CD45−/CD31+ EC population (Figure 4E). Thus, both histological and FACS analyses revealed that RECs are a major source and BMDEPCs is a complementary source of new ECs in endothelial barrier repair in the lungs.

Targeted Inhibition of REC or BMDEPC Intrinsic NF-κB Activity Suppresses REC or BMDEPC Proliferation

We previously demonstrated that doxycycline-induced I-κBαmt expression inhibited NF-κB in endothelial lineage cells but not in other cell types in the EC-I-κBαmt mice.31 Using the EC-I-κBαmt mice as recipients or donors, we generated EC-I-κBα-WT-BM or WT-EC-I-κBα-BM chimeras (Table II in the online-only Data Supplement). We confirmed the high level of donor BM engraftment (>95%) in the chimeras (Figure IB and IC in the online-only Data Supplement). EC-I-κBα-WT-BM or WT-EC-I-κBα-BM mice overexpress I-κBαmt on RECs or BMDEPCs and enabled us to selectively block REC or BMDEPC intrinsic NF-κB activity through doxycycline-induced I-κBαmt expression, which leads to the suppression of REC or BMDEPC proliferation.

At 48 hours post lipopolysaccharide, tissue NF-κB activity is reduced to a low level. Techniques (immunofluorescence and immunohistochemistry) capable of revealing EC-selective NF-κB inhibition are not sensitive enough to detect the subtle change in NF-κB activity caused by doxycycline-induced I-κBmt expression. We verified that treatment of the chimeras with doxycycline induces I-κBαmt expression and inhibits NF-κB activity. We showed that injection of EC-I-κBα-WT-BM mice with doxycycline at 36 hours post lipopolysaccharide induced high level of I-κBαmt mRNA expression (Figure III in the online-only Data Supplement) and repressed lung tissue level of vascular cell adhesion molecule protein, a widely used marker of endothelial NF-κB activity, at 48 hours (Figure 5A).

Figure 5.
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Figure 5.

Targeted suppression of resident endothelial cell (REC) and bone marrow–derived endothelial progenitor cell (BMDEPC) proliferation at barrier repair phase augments endothelial permeability. Wild type (WT), EC-rtTA-GFP-BM (rtTA-GFP), EC-I-κB-WT-BM (I-κB-WT), WT-EC-rtTA-BM (WT-rtTA), and WT-EC-I-κBα-BM (WT-I-κB) mice (Table II in the online-only Data Supplement) were injected with saline (Con) or lipopolysaccharide (LPS; 48 h) and then with doxycycline (Dox; 0.5 mg/mouse IP) 36 hours after LPS injection. At 48 hours after LPS, lung cryosections were prepared or tissue level of vascular cell adhesion molecule (VCAM)-1 protein determined or lung endothelial permeability measured using Evan blue dye (EBD) index. A, Western blot photographs show that Dox-induced I-κBαmt expression at 48 hours reduces the lung tissue level of VCAM-1 protein, a nuclear factor-κB (NF-κB)–dependent gene product, in EC-I-κB-WT-BM mice, indicating an inhibition of REC NF-κB activity. TA-C, EC-rtTA-GFP-BM control, I-κB-C, EC-I-κB-WT-BM control, TA48, EC-rtTA-GFP-BM 48 hours, and I-κB48, EC-I-κB-WT-BM 48 hours. Representative of 3 independent experiments. B and C, Bar graphs show that targeted blockade of REC or BMDEPC intrinsic NF-κB activity suppresses REC or BMDEPC proliferation. Lung cryosections were stained with 5-bromo-2-deoxyuridine (BrdU)+reverse tetracycline transactivator (rtTA) antibodies and nuclei counterstained with DAPI. The number of BrdU+/rtTA+ proliferating RECs (B) or BrdU+/rtTA+ proliferating BMDEPCs (C) was counted and expressed as a percentage of total BrdU+ cells. Mean±SEM of 5 mice per group. *P<0.05, compared with Con groups. #P<0.05, compared with 48-hour group of rtTA-GFP or Wt-rtTA mice. D, Suppression of REC or BMDEPC proliferation by REC- or BMDEPC-targeted NF-κB blockade was associated with an impeded endothelial barrier recovery. Inhibition of REC (white versus black bars) or BMDEPC (white versus grey bars) proliferation was associated with an augmented endothelial permeability. Mean±SEM of 8 mice per group. *P<0.05, compared with Con groups. #P<0.05, compared with WT-48 hour group.

Targeted inhibition of REC or BMDEPC intrinsic NF-κB activity suppressed REC or BMDEPC proliferation. At 48 hours after lipopolysaccharide and 12 hours after doxycycline injection, the number of proliferating RECs or BMDEPCs in lung sections of EC-I-κBα-WT-BM or WT-EC-I-κBα-BM mice (Table II in the online-only Data Supplement, with NF-κB inhibition) was significantly lower than that in lung sections of EC-rtTA-GFP-BM or WT-EC-rtTA-BM mice (Table II in the online-only Data Supplement, without NF-κB inhibition; Figure 5B and 5C).

Suppression of REC or BMDEPC Proliferation at Active Repair Phase Was Associated With an Augmented Endothelial Permeability

At 48 hours after lipopolysaccharide and 12 hours after doxycycline injection, wild-type (WT) mice, in which REC or BMDEPC proliferation was not inhibited, displayed a moderate increase in endothelial permeability that is consistent with endothelial barrier repair phase (Figure 5D). EC-I-κBα-WT-BM or WT-EC-I-κBα-BM mice, in which REC or BMDEPC proliferation was inhibited, exhibited a significantly augmented endothelial permeability (Figure 5D). Blockade of REC intrinsic NF-κB activity caused a 42% reduction in REC proliferation and 41% augmentation of endothelial permeability in the EC-I-κBα-WT-BM mice (Figure 5B versus 5D), suggesting that augmentation of endothelial permeability and suppression of REC proliferation are causally related.

Proliferation-Independent Mechanisms Do Not Contribute to the Augmented Endothelial Permeability Caused by Endothelial NF-κB Blockade at 48 Hours

In addition to controlling EC proliferation, NF-κB mediates endothelial permeability by inducing inflammatory gene expression26 and by disrupting interendothelial junctions, as results of myosin light chain (MLC) phosphorylation and junction protein internalization and downregulation.33–36 To clarify whether these proliferation-independent mechanisms may contribute to the augmented endothelial permeability caused by endothelial NF-κB blockade at 48 hours, we compared lung tissue levels of NF-κB–regulated cytokines, tumor necrosis factor-α, interleukin-1β, interleukin-6, and chemokine (C-X-C motif) ligand 1, between mice at 6 and 48 hours to assess organ inflammatory status at 48 hours, and between WT and EC-I-κBαmt mice to examine the effects of endothelial NF-κB blockade. We used EC-I-κBαmt mice (Table I in the online-only Data Supplement), the recipients or donors of EC-I-κBα-WT-BM or WT-EC-I-κBα-BM chimeras (Table II in the online-only Data Supplement), for these studies. The use of EC-I-κBαmt mice enabled us to evaluate the effects of inhibiting REC and BMDEPC intrinsic NF-κB activities simultaneously. We also compared tissue levels of phospho–MLC-2 and membrane-bound VE-cadherin, 2 major markers of disruption of interendothelial junctions,33–36 between the 2 time points and the 2 groups of mice.

Lung tissue levels of the 4 cytokines at 48 hours were higher than that of controls but were many-fold lower than that at 6 hours, and, importantly, they were not affected by EC-restricted NF-κB inhibition (Figure IVA–IVD in the online-only Data Supplement). At 48 hours post lipopolysaccharide, lung tissue levels of phospho–MLC-2 and membrane-bound and cytoplasmic VE-cadherin proteins were all at control levels and were not affected by EC-restricted NF-κB inhibition (Figure IVE and IVF and Figure V in the online-only Data Supplement). By contrast, the lung tissue level of phospho–MLC-2 was several-fold higher at 3 hours, and the level of membrane-bound or cytoplasimic VE-cadherin protein was several-fold lower or higher at 6 hours than controls and at 48 hours, indicating increased MLC-2 phosphorylation and VE-cadherin internalization at 6 hours but not at 48 hours. All these changes were abrogated by EC-restricted NF-κB blockade (Figure IVE and IVF and Figure V in the online-only Data Supplement). This result excludes the possible involvement of NF-κB–mediated, proliferation-independent mechanisms in the augmentation of endothelial permeability caused by endothelial NF-κB blockade at 48 hours.

Discussion

This study addresses 3 fundamental questions that are critical for the understanding of cellular mechanisms of endothelial barrier restoration after inflammatory organ injury. First, what are the origins of ECs in endothelial barrier repair and restoration? We demonstrated for the first time that both RECs and BMDEPCs are important sources of new ECs in endothelial barrier restoration. The recovery of endothelial barrier function was associated with a remarkably increased REC proliferation and increased BMDEPC proliferation and engraftment. Lungs at active barrier repair phase had the highest level of REC or BMDEPC proliferation and increased BMDEPC engraftment. In lungs, 8 weeks after lipopolysaccharide-induced injury, numbers of REC- and BMDEPC-derived ECs increased by 22- and 121-fold, respectively. More importantly, suppression of REC or BMDEPC proliferation at the active barrier repair phase (48 hours) was associated with an augmented endothelial permeability and impeded endothelial barrier recovery.

Second, what role does each EC precursor cell play in endothelial barrier restoration? We showed that although both RECs and BMDEPCs participate in endothelial barrier repair, their quantitative contributions differ. In lungs at the active barrier repair phase, the number of proliferating RECs was more than double of proliferating BMDEPCs. In lungs, 8 weeks after lipopolysaccharide-induced injury, REC-derived ECs constituted 22%, but BMDEPC-derived ECs constituted only 3.7% of the total CD45−/CD31+ ECs. This result illustrates that REC is a major and BMDEPC is a complementary source of new ECs in endothelial barrier restoration after inflammatory ALI.

RECs and BMDEPCs also play different roles in maintaining normal endothelium. In lungs of mice 8 weeks after saline injection, we detected 1.07% REC-derived new ECs (CD45−/CD31+/rtTA+/BrdU+) but only 0.03% BMDEPC-derived ECs (CD45−/CD31+/eNOS+/GFP+) in the EC population (CD45−/CD31+), suggesting that maintenance of normal endothelium depends mainly on proliferation of RECs with minimal contribution by BMDEPCs. This result is in agreement with a previous report showing that maintenance of lung endothelium does not involve BMDEPCs.15

Third, is there a causal link between REC or BMDEPC proliferation and endothelial barrier restoration? This is the most important question that no previous study has attempted to address because of technical challenges. Cell proliferation is regulated by many signaling pathways and numerous cell cycle regulators with overlapping functions.37,38 The redundancy in the regulatory mechanisms makes it extremely difficult to achieve a clear-cut and high-level inhibition of REC or BMDEPC proliferation by targeting any single proliferation pathway. In addition, many of the proliferation-regulating pathways also regulate other biological processes. This compromises the selectivity in inhibiting cell proliferation. We may not be able to completely overcome the technical huddles using currently available animal models but have to find a way to address this important question. In this study, we used REC- or BMDEPC-restricted inhibition of NF-κB activity as a means of suppressing REC or BMDEPC proliferation. We did so for 3 reasons: first, the NF-κB pathway is a major signaling pathway controlling cell proliferation25–29; second, animal models are available in our laboratory31; and third, inhibition of NF-κB–mediated EC proliferation and inhibition of NF-κB–mediated, proliferation-independent mechanisms have opposite effects on endothelial permeability, which allow us to reliably assess the effect of inhibiting REC or BMDEPC proliferation on endothelial barrier restoration. We found that inhibition of REC or BMDEPC intrinsic NF-κB activity repressed REC or BMDEPC proliferation by 42% or 44%, implicating the important role of NF-κB pathway in controlling REC and BMDEPC proliferation and illustrating the effectiveness of blocking NF-κB pathway in repressing REC or BMDEPC proliferation.

Suppression of REC or BMDEPC proliferation by REC- or BMDEPC-restricted NF-κB blockade at 48 hours was associated with an augmented endothelial permeability. We interpret this augmentation, at least partially, as a consequence of suppressing REC or BMDEPC proliferation. EC apoptosis is a major mechanism underlying the higher endothelial permeability at 48 hours post lipopolysaccharide.30,39 Replacement of the apoptotic/dead ECs through proliferation of endothelial precursor cells (RECs and BMDEPCs) is critical to the restoration of endothelial barrier function. When REC or BMDEPC proliferation is repressed, particularly at the active barrier repair phase, the process of endothelial barrier repair is impeded and endothelial permeability increased. In supporting this contention, we demonstrated here that selective blockade of REC or BMDEPC intrinsic NF-κB activity at 48 hours suppressed REC or BMDEPC proliferation and concomitantly augmented endothelial permeability. Others have demonstrated a causal link between EC apoptosis and increased endothelial permeability.40–42 Our previous study showed that inhibition of EC apoptosis ameliorated the augmentation of endothelial permeability caused by endothelial NF-κB blockade at 48 hours in this ALI model.30,39

In addition to controlling EC proliferation, NF-κB mediates lipopolysaccharide-induced endothelial permeability by inducing inflammatory gene expression,26 which causes the disruption of interendothelial junctions as results of MLC phosphorylation, EC contraction, and junction protein cytosolic translocation.33–36 However, these mechanisms are unlikely to contribute to the augmented endothelial permeability caused by REC- or BMDEPC-targeted NF-κB inhibition at 48 hours. At this stage, tissue levels of NF-κB–regulated cytokines have become low and were not affected by endothelial NF-κB blockade. Tissue levels of phospho-MLC and membrane-bound VE-cadherin, 2 major markers of disruption of interendothelial junctions, were at control levels, suggesting that the lipopolysaccharide-induced disruption of interendothelial junctions has been restored at this time point. More importantly, it is well documented that inhibitions of NF-κB–mediated inflammatory cytokine expression and NF-κB–mediated disruption of interendothelial junctions decrease but not increase endothelial permeability.31,33–36

Other factors may also contribute to the augmentation. NF-κB blockade in BMDEPCs may inhibit BMDEPC migration, adhesion, and incorporation, which can impede BMDEPC-mediated barrier repair. However, this mechanism may not contribute to the augmented endothelial permeability caused by REC-selective NF-κB blockade. Neighboring RECs replace apoptotic/dead ECs by proliferating in situ on the endothelial layer, and by sprouting toward the injured site,6 a process that may not involve significant cell migration and incorporation. To restore endothelial barrier function, the newly generated ECs need to interact with the existing ECs on the endothelial layer to re-establish normal interendothelial junctions. The effects of NF-κB blockade on these cell–cell interactions warrant further investigation. Collectively, these results suggest that suppression of REC or BMDEPC proliferation explains, at least partially, the augmented endothelial permeability caused by blockade of REC or BMDEPC intrinsic NF-κB activity at 48 hours, implying a causal relationship between REC or BMDEPC proliferation and endothelial barrier restoration.

We demonstrated previously that endothelial NF-κB blockade at 6 hours inhibited lipopolysaccharide-induced endothelial permeability31 but showed here that REC- or EPC-targeted NF-κB inhibition at 48 hours augmented endothelial permeability. This discrepancy could be explained by different biological function that NF-κB plays at the 2 time points. At 6 hours, NF-κB mediates the increased endothelial permeability by disrupting interendothelial junctions33–36 without involving significant EC apoptosis.30,39 Endothelial NF-κB blockade at this stage abrogated the disruption of interendothelial junctions but had little effect on EC apoptosis, resulting in a reduced endothelial permeability. At 48 hours, the disrupted interendothelial junctions have been fully restored and EC apoptosis has become a predominant mechanism underlying the increased endothelial permeability. Endothelial NF-κB blockade at this stage inhibited NF-κB–mediated EC proliferation and promoted EC apoptosis,30,39 both of which impeded barrier recovery and augmented endothelial permeability. Consistent with this explanation, we showed here that the tissue level of phospho-MLC or membrane-bound VE-cadherin was remarkably high or low at 6 hours but was at the control level at 48 hours. Endothelial NF-κB blockade abrogated lipopolysaccharide-induced increase in the tissue level of phospho-MLC, decrease in the tissue level of membrane-bound VE-cadherin, and increase in VE-cadherin internalization at 6 hours, but it had no effect at 48 hours. Our previous study showed that endothelial NF-κB blockade at 48 hours enhanced EC apoptosis and concomitantly augmented endothelial permeability.39 Others have demonstrated a causal link between EC apoptosis and increased endothelial permeability.40–42 Although the number of apoptotic/dead ECs is only a small fraction of total ECs, EC apoptosis/death leads to the formation of pores on endothelium, which dramatically increases endothelial leakiness.

Acknowledgments

We thank Dr Amanda Chan, Manager, Feinstein Microscopy Core Facility, and Chris Colon, Director, Feinstein Flow Cytometry Core Facility for their assistance in confocal microscopic image acquisition and analysis, and in fluorescence-activated cell sorting analysis.

Sources of Funding

This work was supported by American Heart Association grant 12GRNT1214002 (S.F. Liu), National Institute of Health grant R21AI076987 (S.F. Liu), and National Nature Science Foundation of China, grant number 81370171 (S.F. Liu).

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.305519/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    ALI
    acute lung injury
    BMDEPCs
    bone marrow–derived endothelial progenitor cells
    BrdU
    bromodeoxyuridine
    EC
    endothelial cell
    eNOS
    endothelial nitric oxide synthase
    FACS
    fluorescence activated cell sorting
    GFP
    green fluorescent protein
    MLC
    myosin light chain
    NF-κB
    nuclear factor-κB
    REC
    resident ECs
    rtTA
    reverse tetracycline transactivator
    WT
    wild-type

  • Received June 24, 2014.
  • Accepted April 29, 2015.
  • © 2015 American Heart Association, Inc.

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Significance

Disruption of endothelial barrier integrity and increase in endothelial leakiness are hallmarks of acute lung injury and other inflammatory conditions. However, the origin and function of new endothelial cells in endothelial barrier repair and restoration are unknown. Using novel transgenic mouse models, this study identifies resident endothelial cells as a major source and bone marrow–derived endothelial progenitor cells as a complementary source of new endothelial cells in endothelial barrier repair and restoration. We provide the first evidence that resident endothelial cells and bone marrow–derived endothelial progenitor cells play important functions in endothelial barrier restoration and demonstrate a causal relationship between resident endothelial cell or bone marrow–derived endothelial progenitor cell proliferation and endothelial barrier restoration. Our data provide important new insights into cellular mechanisms of endothelial barrier repair and restoration.

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Arteriosclerosis, Thrombosis, and Vascular Biology
July 2015, Volume 35, Issue 7
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    Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function After Inflammatory Lung InjurySignificance
    Sun-Zhong Mao, Xiaobing Ye, Gang Liu, Dongmei Song and Shu Fang Liu
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:1635-1644, originally published May 14, 2015
    https://doi.org/10.1161/ATVBAHA.115.305519

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    Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function After Inflammatory Lung InjurySignificance
    Sun-Zhong Mao, Xiaobing Ye, Gang Liu, Dongmei Song and Shu Fang Liu
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:1635-1644, originally published May 14, 2015
    https://doi.org/10.1161/ATVBAHA.115.305519
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