Francisca Pavicic

Kinin B1 receptor activation turns on exocytosis of matrix metalloprotease-9 and myeloperoxidase in human neutrophils: involvement of mitogen-activated protein kinase family

During neutrophil activation and degranulation, MMP‐9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G‐protein‐coupled B1R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP‐9 and MPO by the B1R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin‐treated and ‐untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP‐9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP‐9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B1R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B1R. Our results demonstrate that in human neutrophils, activation of kinin B1R by LDBK initiates separate signaling cascades that trigger the release of MMP‐9 and MPO from tertiary and primary granules, respectively, suggesting that the B1R plays a pivotal role in inflammatory disorders.

Stimulation of the bradykinin B1 receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway

Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B1 (B1R) and B2 receptors. Expression of the B1R in human breast cancer was recently reported, but up to now the consequences of its stimulation are unknown. Our aims were (1) to investigate the capacity of B1R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B1R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B1R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B1R that once stimulated triggered cell proliferation at nanomolar concentrations of the ligand. Inhibitor studies suggested that the proliferative effects depend on the activity of epidermal growth factor receptor and subsequent ERK1/2 mitogen-activated protein kinases phosphorylation. B1R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B1R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B1R may be a valuable alternative for the treatment of breast cancer.

Kinin B1 receptor regulates interactions between neutrophils and endothelial cells by modulating the levels of Mac-1, LFA-1 and intercellular adhesion molecule-1

Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg9]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.

Intracellular signaling pathways involved in the release of IL-4 and VEGF from human keratinocytes by activation of kinin B1 receptor: functional relevance to angiogenesis

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B1 receptor (B1R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B1R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B1R agonist Lys-des[Arg9]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B1R agonist release IL-4 and VEGF. Stimulation of B1R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B1R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2′-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B1R agonist as important angiogenic factors of relevance for skin repair.

Aerogels made of chitosan and chondroitin sulfate at high degree of neutralization: Biological properties toward wound healing: AEROGELS OF CS AND ChS NANOCOMPLEXES

In this study, highly neutralized, highly porous, and ultralight polymeric aerogels prepared from aqueous colloidal suspensions of chitosan (CS) and chondroitin sulfate (ChS) nanocomplexes, formulated as quasi-equimolar amounts of both, are described. These aerogels were designed as healing agents under the inspiration of minimizing the amount of matter applied to wounds, reducing the electrostatic potential of the material and avoiding covalent cross-linkers in order to decrease metabolic stress over wounds. Aerogels synthesized under these criteria are biocompatible and provide specific properties for the induction of wound healing. They do not affect neither the metabolic activity of cultured 3T3 fibroblasts nor the biochemical parameters of experimental animals, open wounds close significantly faster and, unlike control wounds, complete reepithelialization and scarring can be attained 14 days after surgery. Because of its hydration abilities, rapid adaptation to the wound bed and the early accelerator effect of wound closure, the CS/ChS aerogels appear to be functional inducers of the healing. Previous information show that CS/ChS aerogels improve wound bed quality, increase granulation tissue and have pain suppressive effect. CS/ChS aerogels are useful as safe, inexpensive and easy to handle materials for topical applications, such as skin chronic wounds.

Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch‐wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen‐activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up‐regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.