International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors

The calcitonin family of peptides comprises calcitonin, amylin two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

Structure-function analysis of RAMP1 by Alanine Mutagenesis

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

The CGRP1 receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to Gs. R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an α helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. 1290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function. © 2006 American Chemical Society.

Structure-function analysis of RAMP1-RAMP3 chimeras

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

The pharmacology of adrenomedullin 2/intermedin

Adrenomedullin 2 (AM2) or intermedin is a member of the calcitonin gene-related peptide (CGRP)/calcitonin family of peptides and was discovered in 2004. Unlike other members of this family, no unique receptor has yet been identified for it. It is extensively distributed throughout the body. It causes hypotension when given peripherally, but when given into the CNS, it increases blood pressure and causes sympathetic activation. It also increases prolactin release, is anti-diuretic and natriuretic and reduces food intake. Whilst its effects resemble those of AM, it is frequently more potent. Some characterization of AM2 has been done on molecularly defined receptors; the existing data suggest that it preferentially activates the AM receptor formed from calcitonin receptor-like receptor and receptor activity modifying protein 3. On this complex, its potency is generally equivalent to that of AM. There is no known receptor-activity where it is more potent than AM. In tissues and in animals it is frequently antagonised by CGRP and AM antagonists; however, situations exist in which an AM2 response is maintained even in the presence of supramaximal concentrations of these antagonists. Thus, there is a partial mismatch between the pharmacology seen in tissues and that on cloned receptors. The only AM2 antagonists are peptide fragments, and these have limited selectivity. It remains unclear as to whether novel AM2 receptors exist or whether the mismatch in pharmacology can be explained by factors such as metabolism. © 2011 The British Pharmacological Society.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers:opportunities and challenges

Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the 'ADMR'), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.

The activation of the CGRP receptor

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs.

The role of ECL2 in CGRP receptor activation:a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a cal-citonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. © 2013 The Authors.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins

Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute "cell motility" category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the beta- and gamma-proteobacteria. Many fliC genes were deduced to be under the control of sigma(28). The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13-4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (<= 1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.

Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.