IκB kinase activity drives fetal lung macrophage maturation along a non-M1/M2 paradigm.

In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11b(low)F4/80(high) cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms.

Interactions between NF-κB and SP3 connect inflammatory signaling with reduced FGF-10 expression.

Inflammation inhibits normal lung morphogenesis in preterm infants. Soluble inflammatory mediators present in the lungs of patients developing bronchopulmonary dysplasia disrupt expression of multiple genes critical for development. However, the mechanisms linking innate immune signaling and developmental programs are not clear. NF-κB activation inhibits expression of the critical morphogen FGF-10. Here, we show that interactions between the RELA subunit of NF-κB and SP3 suppress SP1-mediated FGF-10 expression. SP3 co-expression reduced SP1-mediated Fgf-10 promoter activity, suggesting antagonistic interactions between SP1 and SP3. Chromatin immunoprecipitation of LPS-treated primary mouse fetal lung mesenchymal cells detected increased interactions between SP3, RELA, and the Fgf-10 promoter. Expression of a constitutively active IκB kinase ß mutant not only decreased Fgf-10 promoter activity but also increased RELA-SP3 nuclear interactions. Expression of a dominant-negative IκB, which blocks NF-κB nuclear translocation, prevented inhibition of FGF-10 by SP3. The inhibitory functions of SP3 required sequences located in the N-terminal region of the protein. These data suggested that inhibition of FGF-10 by inflammatory signaling involves the NF-κB-dependent interactions between RELA, SP3, and the Fgf-10 promoter. NF-κB activation may therefore lead to reduced gene expression by recruiting inhibitory factors to specific gene promoters following exposure to inflammatory stimuli.

Adherens junction proteins in the hamster uterus: their contributions to the success of implantation.

The adherens junction (AJ) is important for maintaining uterine structural integrity, composition of the luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barriers. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin, and alpha-catenin at their mRNA and protein levels, together with the cellular distribution of meprinbeta, phospho-beta-catenin, and active beta-catenin proteins, in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation. By in situ hybridization and immunofluorescence, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. Immunofluorescence study showed no change in epithelial expression patterns of uterine AJ proteins from Days 1 to 5 of pregnancy. With advancement of the implantation process, AJ components were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in the PDZ, but not in the SDZ, of mice. Taken together, these results suggest that AJ proteins contribute to uterine barrier functions by cell-cell adhesion to ensure protection of the embryo. In addition, cleavage of E-cadherin by meprinbeta might contribute to weakening uterine epithelial cell-cell contact for blastocyst implantation. We also report that the nuclear localization of active beta-catenin from Day 4 onward in hamsters implies that beta-catenin/Wnt-signal transduction is activated in the uterus during implantation and decidualization.

Ang-1 gene therapy inhibits hypoxia-inducible factor-1alpha (HIF-1alpha)-prolyl-4-hydroxylase-2, stabilizes HIF-1alpha expression, and normalizes immature vasculature in db/db mice.

OBJECTIVE: Diabetic impaired angiogenesis is associated with impairment of hypoxia-inducible factor-1alpha (HIF-1alpha) as well as vasculature maturation. We investigated the potential roles and intracellular mechanisms of angiopoietin-1 (Ang-1) gene therapy on myocardial HIF-1alpha stabilization and vascular maturation in db/db mice. RESEARCH DESIGN AND METHODS: db/db mice were systemically administrated adenovirus Ang-1 (Ad-CMV-Ang-1). Myocardial HIF-1alpha, vascular endothelial growth factor (VEGF), hemeoxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), Akt, and HIF-1alpha-prolyl-4-hydroxylase-2 (PHD)2 expression were measured. Vasculature maturation, capillary and arteriole densities, and cardiac interstitial fibrosis were analyzed in the border zone of infarcted myocardium. RESULTS: Systemic administration of Ad-CMV-Ang-1 results in overexpression of Ang-1 in db/db mice hearts. Ang-1 gene therapy causes a significant increase in Akt and eNOS expression and HIF-1alpha stabilization. This is accompanied by a significant upregulation of VEGF and HO-1 expression. Intriguingly, Ang-1 gene therapy also leads to a significant inhibition of PHD2 expression. Smooth muscle recruitment and smooth muscle coverage in the neovessels of the border zone of infarcted myocardium are severely impaired in db/db mice compared with wild-type mice. Ang-1 gene therapy rescues these abnormalities, which leads to a dramatic increase in capillary and arteriole densities and a significant reduction of cardiac hypertrophy and interstitial fibrosis at 14 days after ischemia. Taken together, our data show that Ang-1 increases myocardial vascular maturation and angiogenesis together with suppression of PHD2 and the upregulation of HIF-1alpha signaling. CONCLUSIONS: Normalization of immature vasculature by Ang-1 gene therapy may represent a novel therapeutic strategy for treatment of the diabetes-associated impairment of myocardial angiogenesis.

Critical role of the NADPH oxidase subunit p47phox on vascular TLR expression and neointimal lesion formation in high-fat diet-induced obesity.

Reactive oxygen species (ROS) formation is associated with inflammation and vasculature dysfunction. We investigated the potential role of the NADPH oxidase on vascular Toll-like receptor (TLR) expression and carotid neointimal formation in high-fat (HF) diet-induced obesity (DIO) model. Using mice DIO and common carotid artery flow cessation-induced lesion formation models, we examined vascular TLR2 and TLR4 expression and neointimal formation in NADPH oxidase subunit p47(phox)-deficient (p47(phox-/-)) mice. Feeding C57BL/6J mice an HF diet for 22 weeks resulted in significant increases in p47(phox), TLR2 and TLR4 expression in vascular tissues compared with mice fed a low-fat (LF) diet. Minimal changes in TLR2 and TLR4 expression was detected in p47(phox-/-) DIO mice. Furthermore, flow cessation-induced angiogenic and inflammatory response and neointimal formation were significantly attenuated in p47(phox-/-) DIO mice compared with wild-type DIO mice. In addition, exposure of endothelial cells to leptin led to ROS formation; this was accompanied by upregulation of TLR2, TLR4 expression and its downstream signaling. Leptin also increased endothelial cell migration and proliferation. Pharmacological inhibition of NADPH oxidase or genetic deletion of p47(phox) significantly diminished these alterations. Obesity increases neointimal formation via a mechanism involving p47(phox)-TLRs signaling, suggesting that the NADPH oxidase may represent a potential novel therapeutic target for the treatment of obesity-associated vascular inflammation and dysfunction.

Disruption of Ang-1/Tie-2 signaling contributes to the impaired myocardial vascular maturation and angiogenesis in type II diabetic mice.

OBJECTIVE: Microvascular insufficiency represents a major cause of end-organ failure among diabetics. The current studies were undertaken to determine whether dysregulation of the angiopoietins/Tie-2 system would result in an impairment of smooth muscle cell (SMC) recruitment and vascular maturation, which contributes to impaired angiogenesis in diabetes. METHODS AND RESULTS: Tie-2 expression was significantly attenuated, whereas angiopoietin-2 (Ang-2) was increased in db/db mice subjected to myocardial ischemia. Our morphological analysis showed that the number of SMC coverage area per neovessel was significantly reduced in db/db mice. This was accompanied by a significant reduction of myocardial capillary density and arteriole formation. Interestingly, Angiopoietin-1(Ang-1)-induced SMC recruitment and vessel outgrowth were severely impaired in db/db mice. Our in vitro studies further demonstrated that exposure of mouse heart endothelial cells to high glucose resulted in a significant upregulation of Ang-2 and a downregulation of Tie-2 expression. These alterations led to a significant impairment of Ang-1-induced Akt and eNOS phosphorylation, along with a remarkable impairment of Ang-1-induced endothelial cell migration and endothelial cell spheroid sprouting. Ang-1 gene transfer restored Tie-2 expression and rescued these abnormalities in diabetes. CONCLUSIONS: Our findings underscore the important role of Ang-1-Tie-2 signaling in the diabetes-induced impairment of vascular maturation and angiogenesis.

Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are the two ligands of the Tie-2 receptor, a receptor tyrosine kinase that is expressed on the endothelium. A balanced angiopoietin/Tie-2 system is critical for the maintenance of vascular integrity. We investigated the potential role of a disrupted angiopoietin/Tie-2 system on hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Using streptozotocin (STZ) mice subjected to myocardial ischemia, we examined the effects of shifting the Ang-2-to-Ang-1 ratio on myocardial infarction size, apoptosis, bone marrow (BM) cell-endothelial progenitor cell (EPC) differentiation, and angiogenesis. In control mice, myocardial ischemia increased expression of both Ang-2 and Tie-2. In STZ mice, Ang-2 expression was elevated, whereas Tie-2 expression was reduced, and neither was significantly altered by ischemia. Myocardial infarct size and apoptosis were increased in STZ compared with control mice. Using in vivo administration of an adenovirus containing Ang-1 or Ang-2, we found that shifting the Ang-2-to-Ang-1 ratio to favor Ang-1 reduced myocardial apoptosis and infarct size in STZ mice, while shifting the Ang-2-to-Ang-1 ratio to favor Ang-2 resulted in a significant increase in myocardial infarct size and apoptosis in control mice. Myocardial ischemia-stimulated BM cell-EPC differentiation was inhibited and myocardial angiogenesis was reduced in STZ mice. Systemic administration of Ad-Ang-1 restored BM cell-EPC differentiation and increased myocardial VEGF expression and angiogenesis in STZ mice. Our data demonstrate that disturbed angiopoietin/Tie-2 signaling contributes to the hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Restoration of the Ang-2-to-Ang-1 ratio may be a novel therapeutic strategy for the treatment of diabetic myocardial ischemic diseases.