The proangiogenic capacity of polymorphonuclear neutrophils delineated by microarray technique and by measurement of neovascularization in wounded skin of CD18-deficient mice

Growing evidence supports the concept that polymorphonuclear neutrophils (PMN) are critically involved in inflammation-mediated angiogenesis which is important for wound healing and repair. We employed an oligonucleotide microarray technique to gain further insight into the molecular mechanisms underlying the proangiogenic potential of human PMN. In addition to 18 known angiogenesis-relevant genes, we detected the expression of 10 novel genes, namely midkine, erb-B2, ets-1, transforming growth factor receptor-beta(2) and -beta(3), thrombospondin, tissue inhibitor of metalloproteinase 2, ephrin A2, ephrin B2 and restin in human PMN freshly isolated from the circulation. Gene expression was confi rmed by the RT-PCR technique. In vivo evidence for the role of PMN in neovascularization was provided by studying neovascularization in a skin model of wound healing using CD18-deficient mice which lack PMN infi ltration to sites of lesion. In CD18-deficient animals, neo- vascularization was found to be signifi cantly compromised when compared with wild- type control animals which showed profound neovascularization within the granulation tissue during the wound healing process. Thus, PMN infiltration seems to facilitate inflammation mediated angiogenesis which may be a consequence of the broad spectrum of proangiogenic factors expressed by these cells. Copyright (c) 2006 S. Karger AG, Basel

Rapid Accumulation of Polymorphonuclear Neutrophils in the Corpus luteum during Prostaglandin F2α-Induced Luteolysis in the Cow

Prostaglandin F2α (PGF2α) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF2α administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF2α injection. The number of PMNs was increased at 5 min after PGF2α administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF2α directly stimulated P-selectin protein expression at 5–30 min in luteal endothelial cells (LECs). Moreover, PGF2α enhanced PMN adhesion to LECs, and this enhancement by PGF2α was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF2α is crucial in PMN migration. In conclusion, PGF2α rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF2α may involve an acute inflammatory-like response due to rapidly infiltrated PMNs

Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β2 integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class IA. Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class IA as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo

Angiogenesis in Interstitial Lung Diseases: a pathogenetic hallmark or a bystander?

The past ten years parallels have been drawn between the biology of cancer and pulmonary fibrosis. The unremitting recruitment and maintenance of the altered fibroblast phenotype with generation and proliferation of immortal myofibroblasts is reminiscent with the transformation of cancer cells. A hallmark of tumorigenesis is the production of new blood vessels to facilitate tumor growth and mediate organ-specific metastases. On the other hand several chronic fibroproliferative disorders including fibrotic lung diseases are associated with aberrant angiogenesis. Angiogenesis, the process of new blood vessel formation is under strict regulation determined by a dual, yet opposing balance of angiogenic and angiostatic factors that promote or inhibit neovascularization, respectively. While numerous studies have examined so far the interplay between aberrant vascular and matrix remodeling the relative role of angiogenesis in the initiation and/or progression of the fibrotic cascade still remains elusive and controversial. The current article reviews data concerning the pathogenetic role of angiogenesis in the most prevalent and studied members of ILD disease-group such as IIPs and sarcoidosis, presents some of the future perspectives and formulates questions for potential further research

Influence of coating thickness on residual stress and adhesion-strength of cold-sprayed Inconel 718 coatings

In the cold spray process, deposition of particles takes place through intensive plastic deformation upon impact in a solid state at the temperatures well below their melting point. The high particle impact velocity causes high local stresses which lead to deforming the particles and the substrate plastically in the proximity of the particle–substrate interface. As a result, high residual stresses are introduced in cold spray coatings due to the peening effect of the particles collisions with the substrate. In this study, a powder based on the chemical composition of IN 718 was cold-sprayed on IN 718 substrates by using nitrogen gas for an application as a repair tool for aero engine components. The magnitude of the residual stress and its distribution through the thickness were measured by using the hole-drilling and the bending methods. Residual stress was also estimated by using an approach based on the physical process parameters. Mainly compressive residual stresses were observed in cold-sprayed IN 718 coatings. Accumulation of residual stresses in the coatings is highly affected by peening during deposition and it decreases with increase in thickness. It has been observed that the adhesion-strengths of cold-sprayed IN 718 coatings are highly influenced by coating thickness and residual stress states of the coating/substrate system. In the presence of residual stresses in the coatings, adhesion-strength decreases with increasing coating thickness. The energy-release-rate criterion has been used to predict adhesion-strength with increasing coating thickness. Predicted bond-strength values are close to the measured adhesion-strength values and decrease with increase in coating thickness

Influence of coating thickness on residual stress and adhesion-strength of cold-sprayed Inconel 718 coatings

In the cold spray process, deposition of particles takes place through intensive plastic deformation upon impact in a solid state at the temperatures well below their melting point. The high particle impact velocity causes high local stresses which lead to deforming the particles and the substrate plastically in the proximity of the particle–substrate interface. As a result, high residual stresses are introduced in cold spray coatings due to the peening effect of the particles collisions with the substrate. In this study, a powder based on the chemical composition of IN 718 was cold-sprayed on IN 718 substrates by using nitrogen gas for an application as a repair tool for aero engine components. The magnitude of the residual stress and its distribution through the thickness were measured by using the hole-drilling and the bending methods. Residual stress was also estimated by using an approach based on the physical process parameters. Mainly compressive residual stresses were observed in cold-sprayed IN 718 coatings. Accumulation of residual stresses in the coatings is highly affected by peening during deposition and it decreases with increase in thickness. It has been observed that the adhesion-strengths of cold-sprayed IN 718 coatings are highly influenced by coating thickness and residual stress states of the coating/substrate system. In the presence of residual stresses in the coatings, adhesion-strength decreases with increasing coating thickness. The energy-release-rate criterion has been used to predict adhesion-strength with increasing coating thickness. Predicted bond-strength values are close to the measured adhesion-strength values and decrease with increase in coating thickness

Effects of substrate roughness and spray-angle on deposition behavior of cold-sprayed Inconel 718

In this study, Inconel 718 powder particles were successfully cold-sprayed on Inconel 718 substrate by using nitrogen gas for a repair application of aero engine components. The effects of substrate roughness and spray-angle on the deposition behavior of Inconel 718 particles were investigated. It has been found that the deposition behavior of Inconel 718 powder on Inconel 718 substrates is highly influenced by substrate surface roughness. Single powder particle interaction with substrates of different roughness showed that plastic deformation and interfacial material mixing is higher if powder particles interact with substrates of higher roughness. Consequently, substrates with lower roughness demonstrated many abnormalities (e.g. cracks, peeling-off) during the deposition. Substrate roughness is limited to influence the deposition efficiency of the first few layers. Besides, it is also observed that particle deformation and coating quality are significantly affected by the spray-angle because of asymmetric deformation of the particle due to additional tangential momentum. Moreover, it is observed that deposition efficiency is reduced with a decrease in spray-angle, while the coating porosity and coating roughness show an inverse trend. Furthermore, the effects of three input parameters, namely substrate preparation, spray angle and stand-off distance, on four outputs (responses), such as thickness, roughness, porosity and Vickers-hardness of the coating were studied using non-linear statistical regression analysis. The above mentioned four outputs were found to be significantly dependent on substrate preparation and spray-angle

Phosphatidylinositol 3-kinase (PI3K) orchestrates <em>Aspergillus fumigatus</em>-induced eosinophil activation independently of canonical toll-like receptor (TLR)/C-type-lectin receptor (CLR) signaling.

Eosinophilia is associated with various persisting inflammatory diseases and often coincides with chronic fungal infections or fungal allergy as in the case of allergic bronchopulmonary aspergillosis (ABPA). Here, we show that intranasal administration of live Aspergillus fumigatus conidia causes fatal lung damage in eosinophilic interleukin-5 (IL-5)-transgenic mice. To further investigate the activation of eosinophils by A. fumigatus, we established a coculture system of mouse bone marrow-derived eosinophils (BMDE) with different A. fumigatus morphotypes and analyzed the secretion of cytokines, chemokines, and eicosanoids. A. fumigatus-stimulated BMDE upregulated expression of CD11b and downregulated CD62L and CCR3. They further secreted several proinflammatory mediators, including IL-4, IL-13, IL-18, macrophage inflammatory protein-1α (MIP-1α)/CC chemokine ligand 3 (CCL3), MIP-1β/CCL4, and thromboxane. This effect required direct interaction and adherence between eosinophils and A. fumigatus, as A. fumigatus culture supernatants or A. fumigatus mutant strains with impaired adhesion elicited a rather poor eosinophil response. Unexpectedly, canonical Toll-like receptor (TLR) or C-type-lectin receptor (CLR) signaling was largely dispensable, as the absence of MYD88, TRIF, or caspase recruitment domain-containing protein 9 (CARD9) resulted in only minor alterations. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway in A. fumigatus-induced eosinophil activation. Correspondingly, we could show that phosphatidylinositol 3-kinase (PI3K) inhibitors successfully prevent A. fumigatus-induced eosinophil activation. The PI3K pathway in eosinophils may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders. IMPORTANCE Allergic bronchopulmonary aspergillosis (ABPA) is caused by the fungus Aspergillus fumigatus, afflicts about five million patients globally, and is still a noncurable disease. ABPA is associated with pronounced lung eosinophilia. Activated eosinophils enhance the inflammatory response not only by degranulation of toxic proteins but also by secretion of small effector molecules. Receptors and signaling pathways involved in activation of eosinophils by A. fumigatus are currently unknown. Here, we show that A. fumigatus-elicited activation of eosinophils requires direct cell-cell contact and results in modulation of cell surface markers and rapid secretion of cytokines, chemokines, and lipid mediators. Unexpectedly, this activation occurred independently of canonical Toll-like receptor or C-type lectin receptor signaling. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway, and PI3K inhibitors successfully prevented A. fumigatus-induced eosinophil activation. The PI3K pathway may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders