Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development

Abstract Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa‐B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKβ, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKβ demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKβ induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKβ regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKβ regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKβ as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKβ activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.

orchestrate pulmonary angiogenesis during alveolarization remain to be elucidated.
Accumulating evidence indicates that the nuclear factor kappa B (NFjB) signalling pathway plays a key role in both physiologic and pathologic angiogenesis. The NFjB family of transcription factors consists of 5 members each able to bind DNA, and dimerize with other family members. 4 Canonical activation of NFjB occurs by phosphorylation and degradation of IjBs by the IjB kinases, IKKa and IKKb, resulting in rapid translocation of active NFjB complexes into the nucleus to regulate critical genes in cell functions. 5 NFjB enhances angiogenic sprouting during wound healing, and promotes tumour angiogenesis by increasing angiogenic molecules such as vascular endothelial growth factor (VEGF). 6,7 Further, we recently showed that NFjB is constitutively active in the neonatal pulmonary endothelium but quiescent in the adult tissue. 8 Administration of a pharmacologic inhibitor of IKKa and IKKb disrupts alveolarization in neonatal mice, and impairs survival, proliferation and in vitro angiogenesis of primary pulmonary endothelial cells (PECs) isolated from the early alveolar lung. 8 However, the mechanisms allowing temporal-specific activation of NFjB in the pulmonary circulation during alveolarization are not known. IKKa and IKKb have structurally similar catalytic subunits, and both kinases are able to phosphorylate IjB and induce canonical activation of NFjB dimers. 9 However, IKKa and IKKb also possess NFjB-independent functions, allowing unique role for the 2 kinases, and likely accounting for the contradictory results frequently obtained in experimental studies targeting IKKa and IKKb individually. For example, IKKa is increased in the vasculature of lung adenocarcinomas, and over-expression of IKKa, but not IKKb, in HUVECs promotes in vitro angiogenesis. 10 In contrast, homozygous deletion of IKKb in endothelial cells disrupts placental vascularization and impairs EC survival and migration. 11 Taken together, these data suggested that IKKa and IKKb may direct independent functions in the vasculature, with each kinase serving as the predominant regulator of angiogenesis depending on the tissue type and/or stage of development.
Therefore, in this study, we sought to determine the specific roles for IKKa and IKKb in the pulmonary vasculature during postnatal lung development. We performed microarray analysis of gene expression in neonatal PECs after silencing either IKKa or IKKb, and found that the 2 kinases regulate unique panels of genes in the pulmonary vasculature. The loss of either kinase resulted in distinct impairments in physiologic functions essential for angiogenesis including, proliferation, survival, adhesion, migration and cytoskeletal remodelling. Although the number of dysregulated genes was higher in response to silencing IKKa, the functional effects on angiogenic function were substantially greater with loss of IKKb. Taken together, these data suggest that IKKa and IKKb regulate distinct but complementary patterns of genes important for angiogenic function in the pulmonary circulation and identify IKKb as the predominant regulator of angiogenesis in the pulmonary endothelium during postnatal lung development.

| PEC isolation
Neonatal mouse (6 day old, C57BL/6) lungs were digested with collagenase IA (0.5 mg/mL) for 30 minutes at 37°C, and resulted cells were incubated with anti-CD31-coated magnetic beads (Dynabeads, Invitrogen). PECs were isolated by magnetic separation and cultivated as previously described. 8 A description of the characterization of these cells is provided in our previously published manuscript by Iosef et al 8 , where we showed that PEC isolated by this method were found to be exclusively CD45(À) and greater than 95% CD31 (+)/CD102(+) by flow cytometry.

| Microarray
Gene expression patterns were assessed using GeneChip â Prime-View TM Mouse Gene Expression Array (Affymetrix) according to the manufacturer's protocols. Single-stranded cDNA was synthesized from total RNA obtained from PEC transfected with NTC, anti-IKKa or anti-IKKb siRNA and hybridized to gene arrays. Differentially expressed genes were determined by Significance Analysis of Microarray (SAM) method and by R software. Drafting principles were applied by fold change ≥2 or ≤0.5, and q-value ≤5%. Further analysis was then performed with iPathwaysGuide from Advaita Bioinformatics (Plymouth, MI, USA) to assess gene ontology and enriched functional pathways with pathway topologies, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) Ontology Based Annotation System (KOBAS 2.0) database. 12 Comparisons between groups and Meta-Analysis were performed, revealing common and differential gene expression in the 3 groups of PEC.

| Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from PEC and whole mouse lung using the RNeasy kit (Qiagen). RNA (2 lg) was reverse-transcribed using Superscript III (Invitrogen), and qPCR was performed using TaqMan primers (Applied Biosystems) as described previously. 13 Quantification of target gene expression was performed using the standard curve method and normalizing to the expression of either 18s or b2M which were both stable in our transfected cells through experiments performed over several years.

| Apoptosis assays
At 24 hours post-transfection, cells were transferred to 96-well plates (1 9 10 4 cells/well) and after reaching 80%-90% confluence, the media was exchanged for starvation media (FBS 0.2% without additional growth factors). Separate groups of cells were exposed to TNFa (20 ng/mL) to induce apoptosis for 4, 8 and 24 hours. Apoptosis was measured by determining the amount of active caspase 3/7 using a Luciferase-Glo system (Promega). The assays were carried out per manufacturer's protocol, and the luciferase reactions read using a Berthold LG&G Lumat LG luminometer as performed previously. 8

| Western blot
Whole cell lysates were obtained from PEC using a lysis buffer containing 10 mmol/L Tris-HCl and 1% SDS, heated to 100°C. Protein lysates were subjected to SDS-PAGE. Membranes were incubated with primary antibodies to detect Akt, pAKT, p38, ERK1/2 (Cell Signaling), tubulin (Abcam), IKKa and IKKb (Cell Signaling) and the appropriate HRP-conjugated secondary antibody, followed by enhanced chemiluminescence detection (GE Life Sciences).

| Wound healing assay
At 24 hours post-siRNA transfection, PECs were plated on BD-Falcon culture slides coated with 0.2% gelatine. Once confluent, the monolayer was wounded with a fine pipette-tip creating a wound along the diagonal axis of the well. The wound was permitted to heal by PEC migration for the next 24 hours. Phase contrast images were captured before and after the healing process using a Nikon Eclipse inverted microscope with NIS Elements software. The wound closure area was analysed using Image-J software.

| Tube formation assay
Pulmonary endothelial cells were transfected with NTC, IKKb or VCAM siRNA as described above. At 48 hours after transfection,

| Statistics
All data are presented as means AE SE. Statistical differences between 2 groups were determined by Student's t test and between more than 2 groups by either one-way or two-way ANOVA, followed by Bonferroni's multiple-comparison post hoc analysis. A P value of ≤.05 was considered statistically significant. All experiments have been performed with multiple biological and technical replicates, with specific replicate numbers detailed in the figure legends.

| Silencing IKKa and IKKb in PECs dysregulates unique panels of genes
To evaluate the distinct roles for IKKa vs IKKb in neonatal PECs, we disrupted IKK signalling in a subunit-specific manner by RNA interference. Transfection of PEC with siRNA to IKKa decreased IKKa mRNA levels by 82% (P < .0001 vs NTC siRNA, n = 12) without affecting IKKb mRNA or protein levels ( Figure 1A,E). Similarly, transfection of PEC with siRNA to IKKb decreased IKKb mRNA by 89% (P < .0001 vs NTC siRNA, n = 12), without altering IKKa mRNA or protein levels ( Figure 1B and IKKb siRNA and 6 were down-regulated by silencing IKKa, but up-regulated by silencing by IKKb. We then performed functional clustering using the Unified Human Interactome database (UniHI 4), and pathway analyses using i-Pathway-Guide software, and identified enrichment of genes in processes relevant including: cell cycle, proliferation, survival, apoptosis, cell adhesion, cytoskeletal organization, and G-protein coupled signalling, and especially to angiogenesis (Table 1). These analyses identified distinct gene panels regulated by

| Silencing IKKb impairs PEC proliferation in response to selected growth factors
Analyses of the genes differentially regulated by the IKKs revealed multiple factors important in cell cycle checkpoints and proliferation which were altered by silencing IKKa, including cyclin D1 (CCND1), 14 the transcription factor E2F3 15 and Akt3. 16 In contrast, fewer genes relevant to proliferation appeared dysregulated by IKKb silencing but included up-regulation of Flt1, a molecule able to antagonize VEGFmediated angiogenic function. 17 To determine whether IKKa and IKKb mediated regulation of these unique gene sets resulted in differential effects on proliferation, we performed BrdU incorporation assays on the siRNA transfected PEC in response to stimulation with complete endothelial growth media (EGM), or specific individual growth factors.
This data are summarized in Figure 2. NTC siRNA-treated cells proliferated robustly in response to EGM, VEGF or FGF-2, but no proliferative response was observed with IGF-1. IKKa siRNA-treated PECs responded similarly to controls, demonstrating robust proliferation in response to complete EGM, VEGF and FGF-2, but no proliferation in response to IGF-1. In contrast, proliferation was broadly impaired in IKKb siRNA-treated cells, with no significant increase in proliferation in response to EGM, VEGF or FGF-2. Taken together, these data suggest that IKKb plays an indispensable role in promoting neonatal PEC proliferation.

| Silencing IKKa or IKKb impairs PEC survival
The NFjB/IKK signalling pathway can have divergent effects on cell survival, inducing either pro-or anti-apoptotic targets. 18,19 Our prior work demonstrated that simultaneous inhibition of both IKKs with a pharmacologic inhibitor rapidly induces apoptosis in the neonatal PEC. 8 To determine the separate functions of IKKa and IKKb on PEC survival, we measured caspase 3/7 activation in the NTC, IKKa and IKKb siRNA-treated cells. In response to serum withdrawal, apoptosis increased over time for all 3 groups ( Figure 3A), with higher peaks at 8 and 24 hours compared to 4 hours (P < .0001 for NTC siRNA, IKKa siRNA and IKKb siRNA-treated cells). The IKKa T A B L E 1 IKKa and IKKb regulate distinct panels of angiogenic genes in neonatal primary pulmonary endothelial cells. Selected IKKa and IKKb up-or down-regulated genes (>2x) relevant to the main cellular functions of pulmonary endothelial cells and angiogenesis, including motility, cell proliferation and differentiation, survival, and death or turnover

| Silencing IKKa and IKKb induce distinct abnormalities in cell adhesion and motility
In addition to proliferation and survival, effective angiogenesis requires coordinated steps of cell motility, including the attachment of cellular protrusions to the extracellular matrix. 21 Thus, we next evaluated the roles of IKKa and IKKb on mediating cell adhesion to 3 matrices: gelatine, type II collagen and fibronectin. NTC-cells adhered best to collagen ( Figure 4B), and least effectively to gelatine ( Figure 4A). Loss of IKKa disrupted cell adhesion to both collagen and gelatine, but adhesion to fibronectin ( Figure 4C) was preserved.
In contradistinction, loss of IKKb impaired adhesion to all 3 matrices, although adhesion to gelatine was less affected than that observed after the loss of IKKa.
We also evaluated the impact of silencing IKKa, and IKKb on PEC migration by wound-healing assays in the NTC, IKKa and IKKb siRNA-treated neonatal PEC. 22   wound demonstrated prominent central stress fibres, and rapidly organized perpendicular to the short axis of the wound ( Figure 4D).
In contrast, both the IKKa and IKKb siRNA-treated cells demonstrated decreased stress fibre formation and a random disorganization of polarity at the wound edge. Ratio of the wound closure at 24 hours demonstrated that although the loss of either IKKa or IKKb impaired wound healing, loss of IKKb had a more pronounced effect ( Figure 4D).

| Silencing IKKa and IKKb causes distinct defects in actin cytoskeletal rearrangement
Cell migration requires coordinated changes in the remodelling of the actin cytoskeleton to form filopodia, lamellipodia and stress fibres. 21 We next evaluated the effect of silencing either IKKa or IKKb on cytoskeletal remodelling at baseline, and in response to growth factor stimulation. There were no obvious differences in the actin cytoskeleton in the NTC, IKKa and IKKb siRNA-treated PEC under short-term starvation conditions of 12 hours ( Figure 5). We chose to starve the cells for a short term to avoid any apoptosis implication in the process.
In all 3 groups, cortical actin was prominent, with the absence of stress fibres. In the NTC-cells, treatment with either EGM or VEGF induced prominent parallel stress fibres apparent by 5 minutes, and persistent stress fibres at 30 minutes. The IKKa siRNA-treated cells also formed stress fibres in response to both EGM and VEGF at 5 minutes, although the fibres appeared to be slightly fewer in number. By 30 minutes, the stress fibres present in IKKa-depleted cells were appreciably thinner than those observed in control cells. The IKKb siRNA-treated cells stimulated with EGM formed stress fibres that were thinner and less well-aligned than those observed in the NTC- Basal levels of active p38 trended to be higher in the IKKa and IKKb siRNA-treated cell, and they expressed a more modest activation in response to EGM that was not statistically higher than starvation ( Figure 6A). However, mild abnormalities were not observed with the other MAP kinases interrogated, as both IKKa and IKKb silenced cells demonstrated similar patterns of Akt and ERK1/2 activation as compared to control cells ( Figure 6B,C). Representative Western blots to detect phosphorylated and total p38 (A), Akt (B) and ERK (C) 5 min after stimulation with starvation media (0.2% FBS), EGM, or starvation media containing VEGF (50 ng/mL). Data shown are the mean AE SEM of n = 3 immunoblots, with the amount of protein in each band expressed as the fold change over the NTC starvation sample. *P < .05, **P < .01 and ****P < .0001 vs starvation

| IKKb mediated regulation of VCAM serves to promote PEC adhesion and migration
We next sought to identify downstream effector molecules regulated by the NFjB pathway that were promoting angiogenesis in the PEC. Left graph shows the total tube length for each treatment, and the right graph shows the number of branch points. Data shown are mean SEM with n = 3, with **P < .01 vs NTC siRNAtreated cells via one-way ANOVA abnormalities of the PEC during wound closure ( Figure 7E). NTC siRNA-treated PEC located at the leading edge of the wound were organized in parallel, with some cells extending lamellipodia towards the direction of the scratch. In contrast, the VCAM siRNA-treated PEC at the wound edge were disorganized, with many cells extending abnormally long, thin filopodia in multiple directions ( Figure 7E).
To qualify these defects in migration, we compared the effects of silencing VCAM with those induced by silencing IKKb ( Figure 7F) in a wound healing assay. Although the NTC siRNA-treated PEC were able to effectively close the wound in the monolayer, similar impairments in wound healing were observed in response to silencing both IKKb and VCAM. Similarly, using a method to assess VEGF-mediated tube formation, we found that both IKKb and VCAM siRNA-treated PEC developed an overall decrease in the total length of tube formation, and a decreased number of branch points as compared to the NTC siRNA-treated PEC ( Figure 7G).

| DISCUSSION
We previously identified the NFjB as an essential regulator of pulmonary angiogenesis during postnatal lung growth and alveolarization. 8,13 Here we report independent functions for IKKa and IKKb in the developing pulmonary circulation, with IKKb as the predominant regulator of angiogenesis during alveolarization.
In the canonical pathway of NFjB activation, IKKa and IKKb work in concert to release NFjB dimers from the inhibitory IjBs, and allow their translocation to the nucleus to regulate gene expression. Our prior studies found that NFjB is endogenously active in neonatal PECs during early alveolarization, but the mechanism allowing for this developmental activation is not yet clear. Constitutive activity of NFjB is normally restricted to cells from the hematopoietic lineage. 25 In other cell types, basal NFjB activity is negligible and induced only after stimulation. 26 However, high levels of constitutively active NFkB are found in cancer cells, induced by paracrine secretion of cytokines and other factors from the tumour. 27 Therefore, it is possible that secreted factors present in the lung microenvironment allow for temporal-specific activation of the IKKb/NFjB pathway in the developing pulmonary endothelium. Despite significant sequence homology, silencing IKKa and IKKb in PEC resulted in the dysregulation of both shared and unique genes. Both IKKa and IKKb can exert distinct NFjB-dependent and independent effects that vary in a cell stimulus-specific manner. 5 Their separate functions, and limited ability to compensate for each other are highlighted by the distinct embryonic phenotypes resulting from deletion of either IKKa or IKKb. 28,29 Importantly, the IKK complex lacking IKKa is still able to phosphorylate IjB in vitro, suggesting that IKKb is the primary activator of inducible NFjB activation. 30 In addition to NFjB-dependent effects, the IKKs can act differently on transcription factors, 31,32 signalling pathways, 33 mRNA and protein stability, 34,35 chromatin structure. 36 Further studies may determine if NFjB-dependent mechanisms are responsible for inducing the IKKa and IKKb shared targets, and conversely, if NFjB-independent mechanisms are responsible for regulating the IKKa and IKKb unique targets in the developing pulmonary endothelium.
These data add to the work of others associating the IKK/NFjB pathway with physiologic and pathologic angiogenesis. Tie2mediated À/À IKKb phenotype of hematologic and endothelial cells disrupts liver and placental vascular development inducing embryonic lethality, while the AE deletion impairs post-ischaemia neovascularization in adult mice. 11 IKKa is overexpressed in the vasculature of lung adenocarcinoma 10 and increased IKKb has been noted in multiple tumour types. 37 In both cancer and wound healing, the pro-angiogenic effects of the IKK/NFjB pathway have primarily been attributed to the transcriptional regulation of pro-angiogenic cytokines and growth factors. 6,38 In contrast, in both here and in our prior study, we show that IKKa and IKKb have direct effects on PECs, controlling angiogenesis.
In our study, silencing either IKKa or IKKb impaired PEC survival.
Anti-apoptotic functions of the IKK/NFjB pathway have been well described. Deletions of either IKKb or the NFjB subunit RelA result in early embryonic lethality as a result of extensive liver apoptosis. 28 In macrophages, loss of IKKa compromises survival by decreasing Akt phosphorylation. 39 Numerous anti-apoptotic genes are under transcriptional control of the NFjB pathway, including BCL-2 families. 40 NFjB also regulates apoptosis by influencing p53 stability. 41 IKKa too has a separate anti-apoptotic function, influencing the ability of CBP to bind to either p53 or RelA, thus regulating the balance between pro-survival, NFjB signalling and pro-apoptotic p53 signalling. 41,42 We found that IKKb was clearly the predominant regulator of angiogenesis with more profound defects in proliferation, adhesion, migration and cytoskeletal remodelling. In the IKKb depleted cells, proliferation was impaired in response to EGM, VEGF and FGF-2.
Both VEGF and FGF-2 are important growth factors that regulate angiogenesis during development and disease. 43,44 Interestingly, the IKKb depleted cells demonstrated a non-significant trend in increased cell proliferation in response to IGF-1, IGF-1 is expressed in blood vessels of neonatal but not adult animals, and expression is increased in budding capillaries. 45 Blocking the IGF-1 receptor in human and rat foetal lung explants induces endothelial cell loss, increases mesenchymal cell apoptosis and disrupts lung development. 46 This suggests that in the absence of effective VEGF or FGF-2 signalling, compensatory mechanisms through IGF axis may be invoked in the IKKb depleted cells to preserve proliferation.
Vascular endothelial growth factor signalling through the VEGFR2 receptor is an essential pathway mediating postnatal pulmonary angiogenesis during alveolarization. 47,48 VEGF is decreased in the lungs of premature infants dying from BPD and in the lungs of animal models of disrupted alveolarization and pulmonary angiogenesis. 3,[49][50][51] Blocking VEGF/VEGFR2 signalling in neonatal rats decreases pulmonary arterial density and impairs alveolarization, 52 while overexpression of VEGF in newborn rats exposed to hyperoxia preserves pulmonary angiogenesis and alveolarization and increases survival. 53 Loss of IKKb appeared to result in specific defects in VEGF-mediated signalling within the IKKb depleted cells stimulated IOSEF ET AL.

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with VEGF demonstrating severe alterations in actin remodelling, including the absence of stress fibres and increased cortical actin at the periphery. These cytoskeletal abnormalities were still apparent, but milder in the IKKb depleted cells stimulated with EGM, suggesting that additional growth factors in the media may partially compensate for this defect in VEGF signalling. Despite these defects, activation of MAP kinases which promote cell migration, such as p38 were only mildly impaired, suggesting that other effector molecules may be primarily responsible.
We further identified VCAM as one IKKb regulated target that appears to play a significant role in promoting normal PEC adhesion, motility and in vitro angiogenesis ( Figure 7F,G). VCAM-1 is expressed in response to inflammatory cytokines and growth factors and promotes the adherence of leucocytes to activated endothelial cells. 54 However, additional roles for VCAM-1 in embryonic development and pathologic neovascularization have recently been described. Deletion of VCAM-1 induces embryonic lethality between E10.5 and E12.5, resulting in failure of fusion of the allantois to the chorion, and of the endocardium to the myocardium with subsequent cardiac haemorrhage. 55 Moreover, a soluble form of VCAM has also been shown to promote angiogenesis, serving as a chemotactic agent for endothelial cells in inflammatory diseases. 56 To our knowledge, this is the first report suggesting a role for VCAM in promoting physiologic angiogenesis during development. Silencing of VCAM markedly disrupted PEC adhesion and migration, and induced the appearance of abnormal filopodia formation. Endothelial filopodia serve as antennae, detecting chemical and mechanical cues in the environment. Motility is directed by anchoring filopodial protrusions and subsequent retraction of the cell body, moving the cell forward. 57 Further studies will need to explore whether the effect we observed in the VCAM depleted cells is secondary to decreases in soluble VCAM expression, or if cell surface VCAM has a previously unrecognized function in mediating appropriate sensing and anchoring of filopodia.
In addition to VCAM, we found that IKKa and IKKb regulate over 700 genes in the PEC. Although the scope of this study did not permit a comprehensive investigation of all the putative downstream targets that may have a role in mediating angiogenic function in the PEC, there were numerous candidates identified by microarray that could play critical roles. These include targets that are important in regulating discrete steps in cell motility such as Rac3, 58 Rho GTPases (PAK1) 59 and antiangiogenic factors (IGFB5 and FLT-1). 17,60 Further, silencing of IKKa dysregulated more genes than silencing of IKKb, yet the effect of IKKa depletion on PEC angiogenic function was relatively modest. These data suggest that IKKa may be an important regulator of non-angiongenic homoeostatic functions in pulmonary endothelial cells that may warrant further investigation.
There are a number of limitations to our study. First, given that the loss of both IKKa and IKKb also induced impairments in cell survival it is impossible to fully separate the effects of cell death on other measures of angiogic function, including adhesion, migration and in vitro angiogenesis. However, the greater degree of cell death was observed in response to TNF-a stimulation, or in response to prolonged serum starvation. In attempt to limit these confounders as much as possible, we performed these function assays with either no or short periods of starvation, and with assay times that were also short in nature. Second, although our prior work demonstrated that pharmacologic inhibition of both IKKa and IKKb effectively impairs pulmonary angiogenesis and disrupts alveolarizartion in neonatal mice in vivo, 8 definitive evidence for a role for IKKb in promoting pulmonary angiogenesis in vivo using an inducible, cell-specific model allowing EC-specific deletion at the start of alveolarization has not been done.
In summary, we explored the specific roles of IKKa and IKKb in the developing pulmonary endothelium. Silencing either IKKa and IKKb disrupted hundreds of genes, but the panels of genes regulated by the 2 kinases were distinct, suggesting that each kinase has separate functions in the pulmonary endothelium of developing vessels. Additional studies to assess the functional roles of IKKa and IKKb in the pulmonary endothelium demonstrated that IKKb plays a predominant role in promoting a broad array of cellular functions including survival, proliferation, adhesion, migration and actin reorganization. These angiogenic defects were more exaggerated in cells stimulated with VEGF alone, suggesting a specific impairment in VEGF-mediated angiogenesis. VCAM was identified as an IKKb-regulated downstream target, and silencing of VCAM alone resulted in significant impairments in cell adhesion, migration and tube formation, identifying an important, but previously undescribed role for VCAM in pulmonary vascular development. Angiogenesis is essential for alveolarization, the stage of lung development that exponentially increases gas exchange surface area.
Disrupted angiogenesis contributes to many paediatric diseases, including bronchopulmonary dysplasia, and therapeutic strategies to enhance angiogenesis may have potential benefit to these and other lung diseases marked by impaired angiogenesis. Our prior study showed that disrupting IKK-mediated signalling disrupts both angiogenesis and alveolarization. Data from this study identify IKKb as the predominant regulator of angiogenic function in developing pulmonary endothelial cells and suggest that strategies aimed to specifically preserve and enhance IKKb activity may be an important therapeutic strategy to preserve late lung growth.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.