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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 May 14;35(7):1635–1644. doi: 10.1161/ATVBAHA.115.305519

Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function Following Inflammatory Lung Injury

Sun-zhong Mao 1,2,#, Xiaobing Ye 1,#, Gang Liu 1, Dongmei Song 1, Shu Fang Liu 1,2
PMCID: PMC4483164  NIHMSID: NIHMS687671  PMID: 25977568

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 following 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 active barrier repair phase. In lungs 8 weeks after LPS-induced injury, number of REC-derived ECs (CD45−/CD31+/BrdU+/rtTA+) or BMDEPC-derived ECs (CD45−/CD31+/eNOS+/GFP+) increased by 22- or 121-fold. Suppression of REC or BMDEPC proliferation by blocking REC or BMDEPC intrinsic NF-κB at 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 LPS-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 following inflammatory lung injury.

Keywords: Endothelial barrier repair, endothelial cells, NF-κB, multiple organ injury

Introduction

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. Prior 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 following inflammatory organ injury remain unknown.

Resident endothelial cell (REC) is 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 has 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 models,7-13 but play no role in other models.13-16 Microvascular injury associated with septic ALI differs significantly from models used in prior reports in nature and severity. The contribution of BMDEPCs to endothelial barrier repair in septic organ injury is still unclear. Increased EPC mobilization and/or 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 improve 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 prior 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. Additionally, barrier injury and repair are interrelated. Severity of injury determines the extent of repair. It is ideal to inhibit REC or BMDEPC proliferation only at barrier repair phase. Animal model that enables to selectively inhibit REC or BMDEPC proliferation at 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 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 (Dox)-inducible and REC- or BMDEPC-restricted overexpression of a mutant I-κBα (I-κBαmt). By treating these mice with Dox after peak of lung injury, we were able to inhibit REC or BMDEPC proliferation at 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 following 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, Ref. 30). Based on those observations, we defined 0-24 and 24-96 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, Ref 30). We considered 48 hour as active barrier repair phase, and focused our subsequent studies on 48 hours.

Figure 1. Endothelial barrier recovery is associated with increased resident endothelial cell (REC) and bone marrow derived endothelial progenitor (BMDEPC) proliferation.

Figure 1

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A: Endothelial permeability in lungs was measured using Evan blue dye (EBD) leakage index at indicated time (hours) after saline (Con) or LPS (5 mg/kg, i.p.) injection. Means ± SEM of 6 mice per group. *, p< 0.0.5, compared to control.

B to D: Lung cryosections were prepared at indicated time (hours) after LPS and 4 hours after 5-bromo-2-deoxyuridine (BrdU) injection, stained with BrdU plus rtTA (reverse tetracycline transactivator), BrdU plus GFP (green fluorescence protein) or GFP antibodies, and nuclei counterstained with DAPI (4, 6-diamidino-2-phenylindole). 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 to controls.

Endothelial barrier recovery is associated with increased REC and BMDEPC proliferation

If RECs and BMDEPCs play important roles in endothelial barrier repair, number of proliferating RECs or BMDEPCs should increase at 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 (Supplemental Table I and Table II). 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 (BMMNCs) from donors and chimeras 2 months later confirmed that 95% of BM cells in the chimeras were donor BM origin (Supplemental Figure IA). 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 LPS challenge. We found that 99% of the GFP+ cells are CD45−/CD31+/eNOS+ endothelial lineage cells, and less than 1% of them are CD45+/CD31+ hematopoietic cells (Supplemental Figure II). Our finding is consistent with a prior 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 LPS, 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 (Figures. 1A vs 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 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 (Supplemental Table II) were injected with BrdU at 44 hours after LPS injection to label proliferating cells. Lungs were harvested at 48 hours or at 8 weeks after LPS 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 (IF) of lung sections with rtTA or CD31 antibody. We identified proliferating RECs by BrdU and rtTA double IF 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 co-expressed 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 active barrier repair phase

Figure 2. RECs participate in endothelial repair.

Figure 2

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A: RECs proliferate in situ on the endothelial layer at active repair phase. Lung sections from mice 48 hours after LPS injection were stained with antibodies against proliferative marker, BrdU, REC marker, 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 two epithelial layers (blue). Scale bars: A1, A2, A3, A6 and A7, 40 μm; A4 and A5, 8 μm; A8, A9 and A10, 3 μm.

B and C: Fluorescence activated cell sorting (FACS) 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 to saline-injected mice (Con). Mean ± SEM of 5 mice per group. *, p < 0.05, compared with control.

FACS analysis showed that number of REC-derived new ECs (CD45−/CD31+/BrdU+/rtTA+) was approximately 22-fold higher in lungs of EC-rtTA-GFP-BM mice 8 weeks after LPS-induced injury, compared to lungs from mice 8 weeks after saline injection (Figures 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 LPS 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 active repair phase.

Figure 3. BMDEPCs contribute to endothelial repair.

Figure 3

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A: BMDEPCs engraft into microvessel wall. Lung sections from mice 48 hours after LPS injection were stained with antibodies against BMDEPC marker, GFP, 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 co-expression 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: A1, A2, A3, A6 and A7, 40μm; A4, A5, A8, A9 and A10, 8 μm.

B and C: FACS 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 to 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 to control mice.

We quantified the engrafted BMDEPCs (CD45−/CD31+/eNOS+/GFP+) in lungs from mice 8 weeks after saline or LPS injection, a time point when organ inflammation and inflammation-associated BMDEPC recruitment has been subsided. FACS analysis showed that number of CD45−/CD31+/eNOS+/GFP+ cells was approximately 121-fold higher in LPS-injected than in saline-injected lungs (Figures 3B and C), 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 LPS-induced injury. At 48 hours, number of proliferating RECs was 2.4-fold higher than proliferating BMDEPCs in LPS-challenged lungs, although both proliferating RECs and BMDEPCs were remarkably higher than control lungs (Figures 4A and 4B). In lungs 8 weeks after LPS-induced injury, total number of new ECs, defined as CD45−/CD31+/BrdU+ cells, accounted for approximately 2% of total lung cells, which is in agreement with our previous observation that apoptotic ECs accounted for about 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) (Figures 4C and D).

Figure 4. RECs and BMDEPCs contribute differently to endothelial barrier repair.

Figure 4

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A: Representative micrographs of rtTA plus BrdU (RECs) or GFP plus BrdU (BMDEPCs) double staining of lung sections from EC-rtTA-GFP-BM mice 48 hours after saline (Con) or LPS injection. Number of rtTA+/BrdU+ proliferating RECs was significantly higher than number of GFP+/BrdU+ proliferating BMDEPCs (arrows indicated). Scale bars, 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 to BMDEPCs at 48 hours.

C and D: FACS 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 to 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 to engrafted BMDEPCs.

BMDEPCs may replace apoptotic/dead ECs by differentiation.9 We compared number of REC-derived ECs (CD45−/CD31+/BrdU+/rtTA+ cells) to 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 (Supplemental Table II) 8 weeks after LPS 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 is 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 Dox-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 (Supplemental Table II). We confirmed high level of donor BM engraftment (>95%) in the chimeras (Supplemental Figures IB and IC). 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 Dox-induced I-κBαmt expression, which leads to the suppression of REC or BMDEPC proliferation.

At 48 hours post-LPS, tissue NF-κB activity is reduced to a low level. Techniques (IF, IHC) capable of revealing EC-selective NF-κB inhibition are not sensitive enough to detect the subtle change in NF-κB activity caused by Dox-induced I-κBmt expression. We verified that treatment of the chimeras with Dox induces I-κBαmt expression and inhibits NF-κB activity. We showed that injection of EC-I-κBα-WT-BM mice with Dox at 36 hours post-LPS induced high level of I-κBαmt mRNA expression (Supplemental Figure III), and repressed lung tissue level of vascular cell adhesion molecule (VCAM)-1 protein, a widely used marker of endothelial NF-κB activity, at 48 hours (Figure 5A).

Figure 5. Targeted suppression of REC or BMDEPC proliferation at barrier repair phase augments endothelial permeability.

Figure 5

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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 (see Supplemental Table II) were injected with saline (Con) or LPS (48 Hrs), and then with Dox (0.5 mg/mouse, i.p.) 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 EBD index.

A: Western blot photographs show that Dox-induced I-κBαmt expression at 48 hours reduces lung tissue level of VCAM-1 protein, an 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 BrdU plus rtTA antibodies and nuclei counterstained with DAPI. 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 to 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 vs black bars) or BMDEPC (white vs 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 LPS and 12 hours after Dox injection, number of proliferating RECs or BMDEPCs in lung sections of EC-I-κBα-WT-BM or WT-EC-I-κBα-BM mice (Supplemental Table II, with NF-κB inhibition) were significantly lower than in lung sections of EC-rtTA-GFP-BM or WT-EC-rtTA-BM mice (Supplemental Table II, without NF-κB inhibition) (Figures 5B and 5C).

Suppression of REC or BMDEPC proliferation at active repair phase was associated with an augmented endothelial permeability

At 48 hours after LPS and 12 hours after Dox injection, 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 (Figures 5B vs 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 inter-endothelial junctions, as results of myosin light chain (MLC) phosphorylation, and junction protein internalization and down-regulation.33-36 To clarify whether these proliferation-independent mechanisms my 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, TNF-α, IL-1β, IL-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 (Supplemental Table I), the recipients or donors of EC-I-κBα-WT-BM or WT-EC-I-κBα-BM chimeras (Supplemental Table II), 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-MLC2 and membrane-bound VE-cadherin, two major markers of disruption of inter-endothelial junctions33-36, between the two time points and the two groups of mice.

Lung tissue levels of the 4 cytokines at 48 hours were higher than controls, but were many-fold lower than at 6 hours, and, importantly, were not affected by EC-restricted NF-κB inhibition (Supplemental Figures IVA to IVD). At 48 hours post-LPS, lung tissue levels of phospho-MLC2, and membrane-bound and cytoplasmic VE-cadherin proteins were all at control levels, and were not affected by EC-restricted NF-κB inhibition (Supplemental Figures IVE, IVF and V). By contrast, lung tissue level of phospho-MLC2 was several-fold higher at 3 hours, and 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 MLC2 phosphorylation and VE-cadherin internalization at 6 hours, but not at 48 hours. All these changes were abrogated by EC-restricted NF-κB blockade (Supplemental Figures IVE, IVF and V). 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 three fundamental questions that are critical for the understanding of cellular mechanisms of endothelial barrier restoration following 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. 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 LPS-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 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 active barrier repair phase, number of proliferating RECs was more than double of proliferating BMDEPCs. In lungs 8 weeks after LPS-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 following 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 prior 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, due to 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. Additionally, 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 proliferation.25-29 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-LPS.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 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 LPS-induced endothelial permeability by inducing inflammatory gene expression,26 which causes the disruption of inter-endothelial 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 very low and were not affected by endothelial NF-κB blockade. Tissue levels of phospho-MLC and membrane-bound VE-cadherin, two major markers of disruption of inter-endothelial junctions, were at control levels, suggesting that the LPS-induced disruption of inter-endothelial 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 inter-endothelial 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 inter-endothelial 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 LPS-induced endothelial permeability, 31 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 two time points. At 6 hours, NF-κB mediates the increased endothelial permeability by disrupting inter-endothelial junctions33-36 without involving significant EC apoptosis.30, 39 Endothelial NF-κB blockade at this stage abrogated the disruption of inter-endothelial junctions, but had little effect on EC apoptosis, resulting in a reduced endothelial permeability. At 48 hours, the disrupted inter-endothelial 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 tissue level of phospho-MLC or membrane-bound VE-cadherin was remarkably high or low at 6 hours, but was at control level at 48 hours. Endothelial NF-κB blockade abrogated LPS-induced increase in tissue level of phospho-MLC, decrease in tissue level of membrane-bound VE-cadherin and increase in VE-cadherin internalization at 6 hours, but 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 increase endothelial leakiness.

Supplementary Material

Legacy Supplemental File
Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function Following Inflammatory Lung Injury

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 cell (REC) as a major source and bone marrow derived endothelial progenitor cell (BMDEPC) as a complementary source of new ECs in endothelial barrier repair and restoration. We provide the first evidence that RECs and BMDEPCs play important functions in endothelial barrier restoration, and demonstrate a causal relationship between REC or BMDEPC proliferation and endothelial barrier restoration. Our data provides important new insights into cellular mechanisms of endothelial barrier repair and restoration.

Acknowledgments

The authors would like to thank Dr. Amanda Chan, Manager, Feinstein Microscopy Core Facility and Mr. Chris Colon, Director, Feinstein Flow Cytometry Core Facility for their assistance in confocal microscopic image acquisition and analysis, and in FACS analysis.

Sources of Funding:

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

Non-standard Abbreviations and Acronyms

ALI

acute lung injury

Aqu5

aquaporin-5

BMDEPCs

bone marrow-derived endothelial progenitor cells

BMMNCs

bone marrow mononuclear cells

BrdU

bromodeoxyuridine

DAPI

4',6-diamidino-2-phenylindole

Dox

doxycycline

EC

endothelial cell

eNOS

endothelial nitric oxide synthase

EBD

Evans blue dye

EPCs

endothelial progenitor cells

FACS

fluorescence activated cell sorting

GFP

green fluorescent protein

IF

immunofluorescence

Pro-3

TO-PRO-3 dye

VE-cad

VE-cadherin

MLC2

myosin light chain 2

RECs

resident endothelial cells

rtTA

reverse tetracycline transactivator

VCAM-1

vascular cell adhesion molecule-1

Footnotes

Disclosures: None.

References

  • 1.Lee WL, Slutsky AS. Sepsis and endothelial permeability. N Engl J Med. 2010;363:689–691. doi: 10.1056/NEJMcibr1007320. [DOI] [PubMed] [Google Scholar]
  • 2.Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunctions syndrome. Blood. 2003;101:3765–77. doi: 10.1182/blood-2002-06-1887. [DOI] [PubMed] [Google Scholar]
  • 3.Durham RM, Moran JJ, Mazuski JE, Shapiro MJ, Baue AE, Flint LM. Multiple organ failure in trauma patients. J Trauma. 2003;55:608–616. doi: 10.1097/01.TA.0000092378.10660.D1. [DOI] [PubMed] [Google Scholar]
  • 4.Zhao YY, Gao XP, Zhao YD, Mirza MK, Frey RS, Kalinichenko VV, Wang IC, Costa RH, Malik AB. Endothelial cell-restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury. J Clin Invest. 2006;116:2333–2343. doi: 10.1172/JCI27154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mirza MK, Sun Y, Zhao YD, Potula HH, Frey RS, Vogel SM, Malik AB, Zhao YY. FoxM1 regulates re-annealing of endothelial adherens junctions through transcriptional control of beta-catenin expression. J Exp Med. 2010;207:1675–1685. doi: 10.1084/jem.20091857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Itoh Y, Toriumi H, Yamada S, Hoshino H, Suzuki N. Resident endothelial cells surrounding damaged arterial endothelium reendothelialize the lesion. Arterioscler Thromb Vasc Biol. 2010;30:1725–1732. doi: 10.1161/ATVBAHA.110.207365. [DOI] [PubMed] [Google Scholar]
  • 7.Yamada M, Kubo H, Kobayashi S, Ishizawa K, Numasaki M, Ueda S, Suzuki T, Sasaki H. Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury. J Immunol. 2004;172:1266–1272. doi: 10.4049/jimmunol.172.2.1266. [DOI] [PubMed] [Google Scholar]
  • 8.Hohenstein B, Kuo MC, Addabbo F, Yasuda K, Ratliff B, Schwarzenberger C, Eckardt KU, Hugo CP, Goligorsky MS. Enhanced progenitor cell recruitment and endothelial repair after selective endothelial injury of the mouse kidney. Am J Physiol Renal Physiol. 2010;298:F1504–514. doi: 10.1152/ajprenal.00025.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res. 2004;95:343–353. doi: 10.1161/01.RES.0000137877.89448.78. [DOI] [PubMed] [Google Scholar]
  • 10.Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res. 2003;93:783–790. doi: 10.1161/01.RES.0000096651.13001.B4. [DOI] [PubMed] [Google Scholar]
  • 11.Chamoto K, Gibney BC, Lee GS, Lin M, Collings-Simpson D, Voswinckel R, Konerding MA, Tsuda A, Mentzer SJ. CD34+ progenitor to endothelial cell transition in post-pneumonectomy angiogenesis. Am J Respir Cell Mol Biol. 2012;46:283–289. doi: 10.1165/rcmb.2011-0249OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hristov M, Weber C. Endothelial progenitor cells in vascular repair and remodeling. Pharmacol Res. 2008;58:148–151. doi: 10.1016/j.phrs.2008.07.008. [DOI] [PubMed] [Google Scholar]
  • 13.Rafat N, Tönshoff B, Bierhaus A, Beck GC. Endothelial progenitor cells in regeneration after acute lung injury: do they play a role? Am J Respir Cell Mol Biol. 2013;48:399–40. doi: 10.1165/rcmb.2011-0132TR. [DOI] [PubMed] [Google Scholar]
  • 14.Hagensen MK, Vanhoutte PM, Bentzon JF. Arterial endothelial cells: still the craftsmen of regenerated endothelium. Cardiovasc Res. 2012;95:281–289. doi: 10.1093/cvr/cvs182. [DOI] [PubMed] [Google Scholar]
  • 15.Ohle SJ, Anandaiah A, Fabian AJ, Fine A, Kotton DN. Maintenance and repair of the lung endothelium does not involve contributions from marrow-derived endothelial precursor cells. Am J Respir Cell Mol Biol. 2012;47:11–19. doi: 10.1165/rcmb.2011-0180OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Voswinckel R, Ziegelhoeffer T, Heil M, Kostin S, Breier G, Mehling T, Haberberger R, Clauss M, Gaumann A, Schaper W, Seeger W. Circulating vascular progenitor cells do not contribute to compensatory lung growth. Circ Res. 2003;93:372–379. doi: 10.1161/01.RES.0000087643.60150.C2. [DOI] [PubMed] [Google Scholar]
  • 17.Cribbs SK, Sutcliffe DJ, Taylor WR, Rojas M, Easley KA, Tang L, Brigham KL, Martin GS. Circulating endothelial progenitor cells inversely associate with organ dysfunction in sepsis. Intensive Care Med. 2012;38:429–436. doi: 10.1007/s00134-012-2480-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Burnham EL, Taylor WR, Quyyumi AA, Rojas M, Brigham KL, Moss M. Increased circulating endothelial progenitor cells are associated with survival in acute lung injury. Am J Respir Crit Care Med. 2005;172:854–60. doi: 10.1164/rccm.200410-1325OC. [DOI] [PubMed] [Google Scholar]
  • 19.Lam CF, Roan JN, Lee CH, Chang PJ, Huang CC, Liu YC, Jiang MJ, Tsai YC. Transplantation of endothelial progenitor cells improves pulmonary endothelial function and gas exchange in rabbits with endotoxin-induced acute lung injury. Anesth Analg. 2011;112:620–627. doi: 10.1213/ANE.0b013e3182075da4. [DOI] [PubMed] [Google Scholar]
  • 20.Mao M, Wang SN, Lv XJ, Wang Y, Xu JC. Intravenous delivery of bone marrow-derived endothelial progenitor cells improves survival and attenuates lipopolysaccharide-induced lung injury in rats. Shock. 2010;34:196–204. doi: 10.1097/SHK.0b013e3181d49457. [DOI] [PubMed] [Google Scholar]
  • 21.Li B, Cohen A, Hudson TE, Motlagh D, Amrani DL, Duffield JS. Mobilized human hematopoietic stem/progenitor cells promote kidney repair after ischemia/reperfusion injury. Circulation. 2010;121:2211–2220. doi: 10.1161/CIRCULATIONAHA.109.928796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Krasnodembskaya A, Song Y, Fang X, Gupta N, Serikov V, Lee JW, Matthay MA. Antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide LL-37. Stem Cells. 2010;28:2229–2238. doi: 10.1002/stem.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Németh K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K, Robey PG, Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star RA, Mezey E. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15:42–49. doi: 10.1038/nm.1905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Deregibus MC, Cantaluppi V, Calogero R, Lo Iacono M, Tetta C, Biancone L, Bruno S, Bussolati B, Camussi G. Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood. 2007;110:2440–2448. doi: 10.1182/blood-2007-03-078709. [DOI] [PubMed] [Google Scholar]
  • 25.Shishodia S, Aggarwal BB. Nuclear factor-kappaB activation: a question of life or death. J Biochem Mol Biol. 2002;35:28–40. doi: 10.5483/bmbrep.2002.35.1.028. [DOI] [PubMed] [Google Scholar]
  • 26.Liu SF, Malik AB. NF-κB activation as a pathologic mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol. 2006;290:L622–L645. doi: 10.1152/ajplung.00477.2005. [DOI] [PubMed] [Google Scholar]
  • 27.Kisseleva T, Song L, Vorontchikhina M, Feirt N, Kitajewski J, Schindler C. NF-kappaB regulation of endothelial cell function during LPS-induced toxemia and cancer. J Clin Invest. 2006;116:2955–2963. doi: 10.1172/JCI27392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Losef C, Alastalo TP, Hou Y, Chen C, Adams ES, Lyu SC, Cornfield DN, Alvira CM. Inhibiting NF-κB in the developing lung disrupts angiogenesis and alveolarization. Am J Physiol Lung Cell Mol Physiol. 2012;302:L1023–1036. doi: 10.1152/ajplung.00230.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Pfosser A, El-Aouni C, Pfisterer I, Dietz M, Globisch F, Stachel G, Trenkwalder T, Pinkenburg O, Horstkotte J, Hinkel R, Sperandio M, Hatzopoulos AK, Boekstegers P, Bals R, Kupatt C. NF kappaB activation in embryonic endothelial progenitor cells enhances neovascularization via PSGL-1 mediated recruitment: novel role for LL37. Stem Cells. 2010;28:376–85. doi: 10.1002/stem.280. [DOI] [PubMed] [Google Scholar]
  • 30.Liu G, Ye X, Liu SF. Stage-dependent effects of endothelial NF-κB blockade on endothelial apopotosis and proliferation in endotoxemic mice. Am J Respir Crit Care Med. 2011;183:A4194. [Google Scholar]
  • 31.Ye X, Ding D, Zhou Z, Chen G, Liu SF. Divergent roles of endothelial NF-κB in multiple organ injury and bacterial clearance in murine models of sepsis. J Exp Med. 2008;205:1303–1315. doi: 10.1084/jem.20071393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Nowak G, Karrar A, Holmén C, Nava S, Uzunel M, Hultenby K, Sumitran-Holgersson S. Expression of vascular endothelial growth factor receptor-2 or Tie-2 on peripheral blood cells defines functionally competent cell populations capable of reendothelialization. Circulation. 2004;110:3699–707. doi: 10.1161/01.CIR.0000143626.16576.51. [DOI] [PubMed] [Google Scholar]
  • 33.Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev. 2006;86:279–367. doi: 10.1152/physrev.00012.2005. [DOI] [PubMed] [Google Scholar]
  • 34.Shen Q, Rigor RR, Pivetti CD, Wu MH, Yuan SY. Myosin light chain kinase in microvascular endothelial barrier function. Cardiovasc Res. 2010;87:272–80. doi: 10.1093/cvr/cvq144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kouklis P, Konstantoulaki M, Vogel S, Broman M, Malik AB. Cdc42 regulates the restoration of endothelial barrier function. Circ Res. 2004;94:159–66. doi: 10.1161/01.RES.0000110418.38500.31. [DOI] [PubMed] [Google Scholar]
  • 36.Chatterjee A, Snead C, Yetik-Anacak G, Antonova G, Zeng J, Catravas JD. Heat shock protein 90 inhibitors attenuate LPS-induced endothelial hyperpermeability. Am J Physiol Lung Cell Mol Physiol. 2008;294:L755–63. doi: 10.1152/ajplung.00350.2007. [DOI] [PubMed] [Google Scholar]
  • 37.Duronio RJ, Xiong Y. Signaling pathways that control cell proliferation. Cold Spring Harb Perspect Biol. 2013;5:a008904. doi: 10.1101/cshperspect.a008904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 2013;140:3079–93. doi: 10.1242/dev.091744. [DOI] [PubMed] [Google Scholar]
  • 39.Liu G, Ye X, Miller EJ, Liu SF. NF-κB-to-AP-1 switch: a mechanism regulating transition from endothelial barrier injury to repair in endotoxemic mice. Sci Rep. 2014;4:5543. doi: 10.1038/srep05543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lin SJ, Jan KM, Chien S. Role of dying endothelial cells in transendothelial macromolecular transport. Arteriosclerosis. 1990;10:703–709. doi: 10.1161/01.atv.10.5.703. [DOI] [PubMed] [Google Scholar]
  • 41.Childs EW, Tharakan B, Hunter FA, Tinsley JH, Cao X. Apoptotic signaling induces hyperpermeability following hemorrhagic shock. Am J Physiol Heart Circ Physiol. 2007;292:H3179–189. doi: 10.1152/ajpheart.01337.2006. [DOI] [PubMed] [Google Scholar]
  • 42.Childs EW, Tharakan B, Byrge N, Tinsley JH, Hunter FA, Smythe WR. Angiopoietin-1 inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock. Am J Physiol Heart Circ Physiol. 2008;294:H2285–2295. doi: 10.1152/ajpheart.01361.2007. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

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Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function Following Inflammatory Lung Injury
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