Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells

<p>Abstract</p> <p>Background</p> <p>Alveolar epithelial cells are known as progenitor cells for the restoration from the damage in the lung. Calcitonin gene-related peptide (CGRP) has been reported to play an important role in the proliferation of various types of epithelial and endothelial cells. We investigated the effects of CGRP on the proliferation of alveolar epithelial cells <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>A549 cells were cultured in Dulbecco Modified Eagle Medium with 5% fatal bovin serum for 24 hours, then CGRP was added <it>in vitro</it>. The proliferation of DNA synthesis was measured using 5-bromo-2-deoxyuridine, an analog of thymidine, by enzyme-linked immunosorbent assay.</p> <p>As one intracellular response to CGRP, we examined activation of p44/42- extracellular signal-regulated kinase (ERK) pathway by adding CGRP, using western blotting method.</p> <p>Recombinant adenovirus encoding nuclear-targeted-human β-CGRP (rhCGRP) was administered into Male Wister rat (n = 5, 10 weeks old) lungs by intratracheal instillation <it>in vivo</it>. 7 days after the administration of CGRP, rat lungs were harvested and histological findings and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) were evaluated to examine cell proliferation.</p> <p>Results</p> <p><it>In vitro </it>study, CGRP increased the proliferation of A549 cells in a dose and time dependent manner. CGRP8-37 (inhibitor of CGRP receptor) decreased CGRP induced proliferation of DNA synthesis. Phosphorylation of ERK pathway was observed within 15 minutes and peaked in one hour. U0126 (inhibitor of ERK pathway) decreased CGRP induced proliferation of DNA synthesis.<it>In vivo </it>study, histological examination of the lung indicated proliferation of alveolar epithelial cells in the rhCGRP-treated group and the nuclei of alveolar epithelial cells were positive for PCNA immunostaining.</p> <p>Conclusion</p> <p>In this study, we conclude that CGRP stimulates proliferation of human alveolar epithelial cells <it>in vivo </it>and <it>in vitro</it>.</p

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain

Somatostatin Inhibits Cell Migration and Reduces Cell Counts of Human Keratinocytes and Delays Epidermal Wound Healing in an Ex Vivo Wound Model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing

Role of Calcitonin Gene-Related Peptide in Bone Repair after Cyclic Fatigue Loading

Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading.We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP(8-37), for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP(8-37) was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP(8-37) both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP(8-37) administration.CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton

Effects of in vitro hyperthermia on proliferative responses and lymphocyte activity

Fever is induced by both exogenous products like endotoxin, and endogenous cytokines, most notably IL-1 and IL-6, and tumour necrosis factor (TNF). These mediators are believed to interact with the hypothalamus, to induce enhanced body temperature. However, little is known about the biological effects of fever on the function of the immune system. We here report that a 90-min pulse of mild hyperthermia (40°C) induces enhanced proliferation of peripheral blood mononuclear cells (PBMC). This proliferative response was completely inhibited by antibodies to MHC class II, which had no effect on mitogen-induced proliferation of PBMC. The enzyme-linked immunospot (ELISPOT) assay is a sensitive method for detection of single cells secreting antibodies or cytokines. A 90-min pulse of mild hyperthermia (40°C) induced a significantly enhanced immunoglobulin production in PBMC, as determined by ELISPOT, indicating B cell activation. The T cell cytokine pattern both with and without stimulation with hyperthermia differed between individuals. Enhanced interferon-gamma (IFN-γ) secretion was noted at 39–41°C. This IFN-γ response was inhibited by antibodies to MHC class II and thus was MHC class II-restricted and dependent on antigen-presenting cells. None of the individuals tested showed IL-4 response after stimulation with hyperthermia. These findings favour the notion that fever may play an important role in immune responses, and it is possible that fever may act as a physiological adjuvant, with effects on the immune system both in infection and inflammation of other origins

Transplantation of cultured autologous epithelium after meningococcal septicaemia in a 4-year-old with 35% skin loss

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldA 4-year-old girl with scattered full-thickness skin and soft tissue wounds mainly on the extremities and corresponding to roughly 35% of the total body surface area was treated with early excision and transplantation of cultured autologous epidermal grafts. The transplantation was accomplished in one stage, three weeks after admission. The epidermal cells were cultured in Stockholm, Sweden and the time of transportation of the grafts to Reykjavik, Iceland (seven hours) did not seem to affect the quality, as 85% had taken one week after transplantation. The patient healed completely without further transplantation, but a few local skin flaps were needed to cover exposed articular surfaces. The patient was discharged from hospital with stable grafts five weeks after transplantation