Pulmonary IL-1ß expression in early life causes permanent changes in lung structure and function in adulthood.

Chorioamnionitis, mechanical ventilation, oxygen therapy, and postnatal infection promote inflammation in the newborn lung. The long-term consequences of pulmonary inflammation during infancy have not been well characterized. The aim of this study was to examine the impact of inflammation during the late saccular to alveolar stages of lung development on lung structure and function in adulthood. To induce IL-1ß expression in the pulmonary epithelium of mice with a tetracycline-inducible human IL-1ß transgene, doxycycline was administered via intraperitoneal injections to bitransgenic pups and their littermate controls on postnatal days (PN) 0, 0.5, and 1. Lung structure, inflammation, and airway reactivity were studied in adulthood. IL-1ß production in early life resulted in increased numbers of macrophages and neutrophils on PN21, but inflammation subsided by PN42. Permanent changes in alveolar structure, i.e., larger alveoli and thicker alveolar walls, were present from PN21 to PN84. Lack of alveolar septation thus persisted after IL-1ß production and inflammation had ceased. Early IL-1ß production caused goblet cell hyperplasia, enhanced calcium-activated chloride channel 3 (CLCA3) protein expression, and increased airway reactivity in response to methacholine on PN42. Lymphoid follicles were present adjacent to small airways in the lungs of adult bitransgenic mice, and levels of the B cell chemoattractant CXC-motif ligand (CXCL) 13 were elevated in the lungs of bitransgenic mice compared with controls. In conclusion, IL-1ß-induced pulmonary inflammation in early life causes a chronic lung disease in adulthood.

Vascular Endothelial Growth Factor-B Overexpressing Hearts Are Not Protected From Transplant-Associated Ischemia-Reperfusion Injury.

OBJECTIVES: Cardiac vascular endothelial growth factor-B transgene limits myocardial damage in rat infarction models. We investigated whether heart transplant vascular endothelial growth factor-B overexpression protected against ischemia-reperfusion injury. MATERIALS AND METHODS: We transplanted hearts heterotopically from Dark Agouti to Wistar Furth rats. To characterize the role of vascular endothelial growth factor-B in ischemia-reperfusion injury, we transplanted either long-term human vascular endothelial growth factor-B transgene overexpressing hearts from Wistar Furth rats or short-term adeno-associated virus 9-human vascular endothelial growth factor-B-transduced hearts from Dark Agouti rats into Wistar Furth rats. Heart transplants were subjected to 2 hours of cold and 1 hour of warm ex vivo ischemia. Samples were collected 6 hours after reperfusion. RESULTS: Two hours of cold and 1 hour of warm ischemia increased vascular endothelial growth factor-B mRNA levels 2-fold before transplant and 6 hours after reperfusion. Transgenic vascular endothelial growth factor-B overexpression caused mild cardiac hypertrophy and elevated cardiac troponin T levels 6 hours after reperfusion. Laser Doppler measurements indicated impaired epicardial tissue perfusion in these transgenic transplants. Recombinant human vascular endothelial growth factor-B increased mRNA levels of cytochrome c oxidase and extracellular ATPase CD39, suggesting active oxidative phosphorylation and high ATP production. Adeno-associated virus 9-mediated vascular endothelial growth factor-B overexpression in transplanted hearts increased intragraft macrophages 1.5-fold and proinflammatory cytokine interleukin 12 p35 mRNA 1.6-fold, without affecting recipient serum cardiac troponin T concentration. CONCLUSIONS: Vascular endothelial growth factor-B expression in transplanted hearts is linked to ischemia and ischemia-reperfusion injury. Cardiac transgenic vascular endothelial growth factor-B overexpression failed to protect heart transplants from ischemia-reperfusion injury.

Communication skills training enhances nurses' ability to respond with empathy to parents' emotions in a neonatal intensive care unit.

AIM: We quantitatively analysed the effect of a course in communication on the content of nurse-parent encounters and the ability of nurses to respond to the empathic needs of parents in a level III neonatal intensive care unit. METHODS: We evaluated 36 and 45 nurse-parent encounters audio recorded before and after 13 neonatal nurses attended a communication course. The number of empathic opportunities, the nurses' responses to these and the ways they involved parents in their infants' care were studied. RESULTS: Both before and after the course, the nurses talked more than the parents during the conversations. This nurse-centredness decreased after the course. The use of empathic or exploring responses to empathic opportunities increased from 19.9 ± 9.0% to 53.8 ± 8.9% (p = 0.027), whereas ignoring the feelings of the parents or giving inadequate advice decreased from 63.0 ± 10.0% to 27.5 ± 8.4% (p = 0.043) after the course. Use of statements expressing caring for the parents and encouragement for parents to participate in the care of their infant increased after the course (p = 0.0034 and p = 0.043, respectively). The nurses felt the course was very useful for their profession. CONCLUSION: A course in communication techniques improved nurses' ability to respond to parents' feelings with empathy.

Vascular endothelial growth factor-B in physiology and disease.

Vascular endothelial growth factor-B (VEGF-B), discovered over 15 years ago, has long been seen as one of the more ambiguous members of the VEGF family. VEGF-B is produced as two isoforms: one that binds strongly to heparan sulfate in the pericellular matrix and a soluble form that can acquire binding via proteolytic processing. Both forms of VEGF-B bind to VEGF-receptor 1 (VEGFR-1) and the neuropilin-1 (NRP-1) coreceptor, which are expressed mainly in blood vascular endothelial cells. VEGF-B-deficient mice and rats are viable without any overt phenotype, and the ability of VEGF-B to induce angiogenesis in most tissues is weak. This has been a puzzle, as the related placenta growth factor (PlGF) binds to the same receptors and induces angiogenesis and arteriogenesis in a variety of tissues. However, it seems that VEGF-B is a vascular growth factor that is more tissue specific and can have trophic and metabolic effects, and its binding to VEGFR-1 shows subtle but important differences compared with that of PlGF. VEGF-B has the potential to induce coronary vessel growth and cardiac hypertrophy, which can protect the heart from ischemic damage as well as heart failure. In addition, VEGF-B is abundantly expressed in tissues with highly active energy metabolism, where it could support significant metabolic functions. VEGF-B also has a role in neuroprotection, but unlike other members of the VEGF family, it does not have a clear role in tumor progression. Here we review what is hitherto known about the functions of this growth factor in physiology and disease.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart.

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

IL-1ß expression in the distal lung epithelium disrupts lung morphogenesis and epithelial cell differentiation in fetal mice.

Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1ß in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1ß under the control of the surfactant protein (SP)-C promoter. IL-1ß-expressing pups suffered respiratory failure and died shortly after birth. IL-1ß caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3, the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1ß decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1ß inhibited the expression of VEGF-A and its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1ß-expressing fetal mice. IL-1ß suppressed the production of SP-B and pro-SP-C and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1ß prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1ß. In conclusion, expression of IL-1ß in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.

Immune cells control skin lymphatic electrolyte homeostasis and blood pressure.

The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

VEGF-C and VEGF-D blockade inhibits inflammatory skin carcinogenesis.

VEGF-C and VEGF-D were identified as lymphangiogenic growth factors and later shown to promote tumor metastasis, but their effects on carcinogenesis are poorly understood. Here, we have studied the effects of VEGF-C and VEGF-D on tumor development in the murine multistep chemical carcinogenesis model of squamous cell carcinoma by using a soluble VEGF-C/VEGF-D inhibitor. After topical treatment with a tumor initiator and repeated tumor promoter applications, transgenic mice expressing a soluble VEGF-C/VEGF-D receptor (sVEGFR-3) in the skin developed significantly fewer squamous cell tumors with a delayed onset when compared with wild-type mice or mice expressing sVEGFR-3 lacking the ligand-binding site. Epidermal proliferation was reduced in the carcinogen-treated transgenic skin, whereas epidermal keratinocyte proliferation in vitro was not affected by VEGF-C or VEGF-D, indicating indirect effects of sVEGFR-3 expression. Importantly, transgenic mouse skin was less sensitive to tumor promoter-induced inflammation, with reduced angiogenesis and blood vessel leakage. Cutaneous leukocytes, especially macrophages, were reduced in transgenic skin without major changes in macrophage polarization or blood monocyte numbers. Several macrophage-associated cytokines were also reduced in transgenic papillomas, although the dermal macrophages themselves did not express VEGFR-3. These findings indicate that VEGF-C/VEGF-D are involved in shaping the inflammatory tumor microenvironment that regulates early tumor progression. Our results support the use of VEGF-C/VEGF-D-blocking agents not only to inhibit metastatic progression, but also during the early stages of tumor growth.

Mouse models for studying angiogenesis and lymphangiogenesis in cancer.

The formation of new blood vessels (angiogenesis) is required for the growth of most tumors. The tumor microenvironment also induces lymphangiogenic factors that promote metastatic spread. Anti-angiogenic therapy targets the mechanisms behind the growth of the tumor vasculature. During the past two decades, several strategies targeting blood and lymphatic vessels in tumors have been developed. The blocking of vascular endothelial growth factor (VEGF)/VEGF receptor-2 (VEGFR-2) signaling has proven effective for inhibition of tumor angiogenesis and growth, and inhibitors of VEGF-C/VEGFR-3 involved in lymphangiogenesis have recently entered clinical trials. However, thus far anti-angiogenic treatments have been less effective in humans than predicted on the basis of pre-clinical tests in mice. Intrinsic and induced resistance against anti-angiogenesis occurs in patients, and thus far the clinical benefit of the treatments has been limited to modest improvements in overall survival in selected tumor types. Our current knowledge of tumor angiogenesis is based mainly on experiments performed in tumor-transplanted mice, and it has become evident that these models are not representative of human cancer. For an improved understanding, angiogenesis research needs models that better recapitulate the multistep tumorigenesis of human cancers, from the initial genetic insults in single cells to malignant progression in a proper tissue environment. To improve anti-angiogenic therapies in cancer patients, it is necessary to identify additional molecular targets important for tumor angiogenesis, and to get mechanistic insight into their interactions for eventual combinatorial targeting. The recent development of techniques for manipulating the mammalian genome in a precise and predictable manner has opened up new possibilities for the generation of more reliable models of human cancer that are essential for the testing of new therapeutic strategies. In addition, new imaging modalities that permit visualization of the entire mouse tumor vasculature down to the resolution of single capillaries have been developed in pre-clinical models and will likely benefit clinical imaging.

Transgenic overexpression of interleukin-1ß induces persistent lymphangiogenesis but not angiogenesis in mouse airways.

These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1ß mice that conditionally overexpress IL-1ß in Clara cells to determine whether IL-1ß can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1ß production, influx of neutrophils and macrophages, and conspicuous and persistent lymphangiogenesis, but surprisingly no angiogenesis. Gene profiling showed many up-regulated genes, including chemokines (Cxcl1, Ccl7), cytokines (tumor necrosis factor α, IL-1ß, and lymphotoxin-ß), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1ß overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1ß induced lymphangiogenesis. Inactivation of vascular endothelial growth factor (VEGF)-C and VEGF-D by adeno-associated viral vector-mediated soluble VEGFR-3 (VEGF-C/D Trap) completely blocked lymphangiogenesis, showing its dependence on VEGFR-3 ligands. Consistent with this mechanism, VEGF-C immunoreactivity was present in some Aif1/Iba1-immunoreactive macrophages. Because neutrophils contribute to IL-1ß-induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1ß/CXCR2(-/-) mice had the usual IL-1ß-mediated lymphangiogenesis but no neutrophil recruitment, suggesting that neutrophils are not essential. IL1R1 immunoreactivity was found on some epithelial basal cells and neuroendocrine cells, suggesting that these cells are targets of IL-1ß, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1ß overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.

Role of CXC chemokine receptor-2 in a murine model of bronchopulmonary dysplasia.

The contribution of neutrophils and CXC chemokines to the pathogenesis of bronchopulmonary dysplasia is not well defined. The transgenic expression of IL-1ß in the pulmonary epithelium causes lung inflammation and disrupts alveolar development in infant mice. To study the hypothesis that CXC chemokine receptor-2 (CXCR2) is a mediator of inflammatory lung injury, we compared lung development in IL-1ß-expressing mice with wild-type (IL-1ß/CXCR2(+/+)) or null (IL-1ß/CXCR2(-/-)) CXCR2 loci. CXCR2 deficiency abolished the transmigration of neutrophils into the alveolar lumen in IL-1ß-expressing mice, but did not alter the number of neutrophils in the parenchyma. The deletion of CXCR2 increased the alveolar chord length and reduced the survival of mice when IL-1ß was expressed from the pseudoglandular to the alveolar stages. The capillary configuration was highly abnormal in both IL-1ß/CXCR2(+/+) and IL-1ß/CXCR2(-/-) lungs, but in very different ways. The cellular area of the parenchyma and the total capillary area of IL-1ß/CXCR2(+/+) and IL-1ß/CXCR2(-/-) mice were smaller than those of control/CXCR2(+/+) and control/CXCR2(-/-) mice, but the ratio of capillary area to cellular area was similar in all four genotypes. When IL-1ß was expressed during the saccular stage, IL-1ß/CXCR2(-/-) mice had smaller alveolar chord lengths and better survival than did IL-1ß/CXCR2(+/+) mice. Independent of the timing of IL-1ß expression, IL-1ß increased alveolar septal thickness in mice with wild-type CXCR2 loci, but not in CXCR2 null mice. Depending on the developmental stage at the time of the inflammatory insult, inhibition of the CXCR2 pathway may exert opposite effects on alveolar septation in the neonatal lung.

Perspectives on lymphangiogenesis and angiogenesis in cancer.

Tumor-associated neovascularization allows tumor cells to express their critical growth advantage, whereas lymphatic invasion is crucial for the metastatic process. Various growth factors stimulate blood and lymphatic neovascularization and modulate vessel permeability in tumors. The first anti-angiogenic drugs are already in routine use, and new anti-vascular therapeutics are evaluated in clinical trials. Conversely, pro-lymphangiogenic therapy could be implemented to treat cancer survivors suffering from secondary lymphedema.

Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation.

BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. METHODS AND RESULTS: Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B(167) isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. CONCLUSIONS: VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B-induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B-induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease.

How do angiopoietins Tie in with vascular endothelial growth factors?

PURPOSE OF REVIEW: The endothelial cells of the blood and lymphatic vessels are involved in common human diseases. Excess blood and lymphatic vessel growth enhances tumor progression and metastasis, whereas insufficient growth leads to tissue ischemia and lymphedema. Lymphatic and blood vascular endothelial cells are regulated by two endothelial specific receptor tyrosine kinase systems, the vascular endothelial growth factor (VEGF) receptors (VEGFRs) and the Tie receptors, activated by the VEGF and angiopoietin ligands, respectively. Blocking of the VEGF-VEGFR-2 pathway has provided the first antiangiogenic strategy for cancer therapy and here we discuss the other pathways where progress is made for drug development, in particular the angiopoietin (Ang)-Tie receptor pathway. RECENT FINDINGS: VEGF-activated VEGFR-2 is the major transducer of angiogenic signals, but recent results show that the lymphangiogenic VEGFR-3 induces angiogenic sprouting as well. VEGF-B, a member of the VEGF family with low angiogenic activity, has been found to be involved in the regulation of energy metabolism in the heart. Recent reports have implicated Ang2 in tumor angiogenesis and revealed that Tie2 utilizes a unique signaling mechanism at endothelial cell-cell junctions. SUMMARY: The VEGF-VEGFR and Ang-Tie systems regulate different aspects of blood and lymphatic vessel growth. Thus, targeting both systems may be beneficial in maximizing the efficacy of anti/pro-angiogenic therapies.

VEGFs and receptors involved in angiogenesis versus lymphangiogenesis.

Vascular endothelial growth factors and their endothelial tyrosine kinase receptors are central regulators of vasculogenesis, angiogenesis and lymphangiogenesis. VEGF signalling through VEGFR-2 is the major angiogenic pathway, and blockage of VEGF/VEGFR-2 signalling is the first anti-angiogenic strategy for cancer therapy. VEGFR-1 seems to act as a negative regulator of VEGF-mediated angiogenesis during development, and as a stimulator of pathological angiogenesis when activated by its specific ligands PlGF and VEGF-B. PlGF recruits angiogenic macrophages to tumours, and targeting PlGF could therefore be beneficial in cancer. For VEGF-B, with very limited angiogenic potential, a new role has been identified in regulating lipid metabolism in the heart. VEGF-C and VEGF-D induce lymphangiogenesis via VEGFR-3 and have also been shown to be lymphangiogenic in tumours, stimulating metastasis. Mouse models of lymphoedema have established VEGF-C as a promising agent for pro-lymphangiogenic therapy. In addition to lymphangiogenesis, VEGFR-3 has also been shown to be important for angiogenesis, acting together with VEGF/VEGFR-2 and Dll4/Notch signalling to control angiogenic sprouting. Increasing knowledge of the mechanisms regulating (lymph)angiogenesis should enable the development of better agents to combat metastasis and the resistance of tumours towards anti-angiogenic treatment, and of pro-(lymph)angiogenic treatment methods for ischaemic diseases and lymphoedema.

Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy.

Vascular endothelial growth factor (VEGF)-B is poorly angiogenic but prominently expressed in metabolically highly active tissues, including the heart. We produced mice expressing a cardiac-specific VEGF-B transgene via the alpha-myosin heavy chain promoter. Surprisingly, the hearts of the VEGF-B transgenic mice showed concentric cardiac hypertrophy without significant changes in heart function. The cardiac hypertrophy was attributable to an increased size of the cardiomyocytes. Blood capillary size was increased, whereas the number of blood vessels per cell nucleus remained unchanged. Despite the cardiac hypertrophy, the transgenic mice had lower heart rate and blood pressure than their littermates, and they responded similarly to angiotensin II-induced hypertension, confirming that the hypertrophy does not compromise heart function. Interestingly, the isolated transgenic hearts had less cardiomyocyte damage after ischemia. Significantly increased ceramide and decreased triglyceride levels were found in the transgenic hearts. This was associated with structural changes and eventual lysis of mitochondria, resulting in accumulation of intracellular vacuoles in cardiomyocytes and increased death of the transgenic mice, apparently because of mitochondrial lipotoxicity in the heart. These results suggest that VEGF-B regulates lipid metabolism, an unexpected function for an angiogenic growth factor.