Multidrug resistance proteins preferentially regulate natriuretic peptide-driven cGMP signalling in the heart and vasculature.

BACKGROUND AND PURPOSE: cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP-driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. EXPERIMENTAL APPROACH: Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload-induced heart failure was also explored. KEY RESULTS: MK571 attenuated hCASMC growth and enhanced the anti-proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration-dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

The Zebrafish Cardiac Endothelial Cell-Roles in Development and Regeneration.

In zebrafish, the spatiotemporal development of the vascular system is well described due to its stereotypical nature. However, the cellular and molecular mechanisms orchestrating post-embryonic vascular development, the maintenance of vascular homeostasis, or how coronary vessels integrate into the growing heart are less well studied. In the context of cardiac regeneration, the central cellular mechanism by which the heart regenerates a fully functional myocardium relies on the proliferation of pre-existing cardiomyocytes; the epicardium and the endocardium are also known to play key roles in the regenerative process. Remarkably, revascularisation of the injured tissue occurs within a few hours after cardiac damage, thus generating a vascular network acting as a scaffold for the regenerating myocardium. The activation of the endocardium leads to the secretion of cytokines, further supporting the proliferation of the cardiomyocytes. Although epicardium, endocardium, and myocardium interact with each other to orchestrate heart development and regeneration, in this review, we focus on recent advances in the understanding of the development of the endocardium and the coronary vasculature in zebrafish as well as their pivotal roles in the heart regeneration process.

Neuropilin 1 mediates epicardial activation and revascularization in the regenerating zebrafish heart.

Unlike adult mammals, zebrafish can regenerate their heart. A key mechanism for regeneration is the activation of the epicardium, leading to the establishment of a supporting scaffold for new cardiomyocytes, angiogenesis and cytokine secretion. Neuropilins are co-receptors that mediate signaling of kinase receptors for cytokines with crucial roles in zebrafish heart regeneration. We investigated the role of neuropilins in response to cardiac injury and heart regeneration. All four neuropilin isoforms (nrp1a, nrp1b, nrp2a and nrp2b) were upregulated by the activated epicardium and an nrp1a-knockout mutant showed a significant delay in heart regeneration and displayed persistent collagen deposition. The regenerating hearts of nrp1a mutants were less vascularized, and epicardial-derived cell migration and re-expression of the developmental gene wt1b was impaired. Moreover, cryoinjury-induced activation and migration of epicardial cells in heart explants were reduced in nrp1a mutants. These results identify a key role for Nrp1 in zebrafish heart regeneration, mediated through epicardial activation, migration and revascularization.

Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

Neuropilins 1 and 2 mediate neointimal hyperplasia and re-endothelialization following arterial injury.

AIMS: Neuropilins 1 and 2 (NRP1 and NRP2) play crucial roles in endothelial cell migration contributing to angiogenesis and vascular development. Both NRPs are also expressed by cultured vascular smooth muscle cells (VSMCs) and are implicated in VSMC migration stimulated by PDGF-BB, but it is unknown whether NRPs are relevant for VSMC function in vivo. We investigated the role of NRPs in the rat carotid balloon injury model, in which endothelial denudation and arterial stretch induce neointimal hyperplasia involving VSMC migration and proliferation. METHODS AND RESULTS: NRP1 and NRP2 mRNAs and proteins increased significantly following arterial injury, and immunofluorescent staining revealed neointimal NRP expression. Down-regulation of NRP1 and NRP2 using shRNA significantly reduced neointimal hyperplasia following injury. Furthermore, inhibition of NRP1 by adenovirally overexpressing a loss-of-function NRP1 mutant lacking the cytoplasmic domain (ΔC) reduced neointimal hyperplasia, whereas wild-type (WT) NRP1 had no effect. NRP-targeted shRNAs impaired, while overexpression of NRP1 WT and NRP1 ΔC enhanced, arterial re-endothelialization 14 days after injury. Knockdown of either NRP1 or NRP2 inhibited PDGF-BB-induced rat VSMC migration, whereas knockdown of NRP2, but not NRP1, reduced proliferation of cultured rat VSMC and neointimal VSMC in vivo. NRP knockdown also reduced the phosphorylation of PDGFα and PDGFß receptors in rat VSMC, which mediate VSMC migration and proliferation. CONCLUSION: NRP1 and NRP2 play important roles in the regulation of neointimal hyperplasia in vivo by modulating VSMC migration (via NRP1 and NRP2) and proliferation (via NRP2), independently of the role of NRPs in re-endothelialization.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle.

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K(+), suggesting that it is regulated not only by K(+) conductances but by inward conductances such as Na(+) and/or Ca(2+). We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na(+) with equimolar tetraethylammonium or Ca(2+) with Mn(2+) inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na(+) with N-methyl-D-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bI(NSCC)) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bI(NSCC). In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.

Molecular detection of Hematodinium sp. in Northeast Pacific Chionoecetes spp. and evidence of two species in the Northern Hemisphere.

Hematodinium is a genus of parasitic dinoflagellates that infects crustaceans worldwide including Tanner crabs Chionoecetes bairdi and snow crabs C. opilio in the Northeast Pacific Ocean. The present study describes the optimization of a PCR-based assay for the detection and monitoring of Hematodinium sp. in snow and Tanner crabs. Two fragments, 1682 and 187 bp, were amplified from the 18S ribosomal DNA region of the parasite. The assay performed well in 6 additional decapod species (1 lobster and 5 crabs) infected with Hematodinium spp., suggesting that it could be used to detect Hematodinium spp. in other decapods. We also report Hematodinium spp. infections in the majid crab, Hyas coarctatus, and the lithodid crab, Lithodes couesi. Sequencing of 18S rDNA and the adjacent internal transcribed spacer 1 (ITS1) region of Hematodinium spp. isolated from 7 host species in the present study revealed the presence of 2 Hematodinium clades, one in the blue crab Callinectes sapidus and a second in all other host species. The ITS1 sequences of the 2 clades could not be aligned, but showed a conserved secondary structure that may be related to a functional diversification during a host switch. Comparison of our data with 18S and ITS1 sequence data available in GenBank placed the north Pacific Hematodinium sp. in a clade separate from the Hematodinium sp. infecting the portunoids, C. sapidus, Liocarcinus depurator and Scylla serrata, and within a second clade that infected all other decapod hosts located in the North Pacific and North Atlantic Oceans.

Large variations occur in bone density measurements of children when using different software.

BACKGROUND: Paediatric dual X-ray absorptiometry (DXA) studies present a number of technical problems. One of these is that the edge detection algorithms designed for the adult skeleton may fail for paediatric studies. Hologic provide alternative algorithms for low bone density studies. AIM: To assess low-density software for the analysis of paediatric DXA studies and to compare with the adult protocol. METHODS: Our centre has scanned 450 normal children as part of a normal range study. A subgroup of 103 children was selected using a random number generator. The group was distributed evenly between males and females and across the age range 5-17 years. Each individual underwent both a lumbar spine and a whole-body scan on a Hologic QDR-4500W DXA scanner. Both scans were analysed using the standard adult protocol and then re-analysed using the Hologic experimental paediatric protocol for whole body and the Hologic low-density protocol for lumbar spine. RESULTS: Both lumbar spine protocols showed an increase in bone mineral density with age; however, the low-density protocol always produced a lower bone mineral density result than the adult protocol. Bland-Altman analysis showed limits of agreement of 0.031-0.093 g x cm(-2) (male, 0.032-0.089 g x cm(-2); female, 0.031-0.096 g x cm(-2)). This represents a mean difference of 9%. Five results showed differences greater than the upper limit of agreement. All these cases were children under 11 years of age who had large areas of spine not identified as bone by the adult protocol. These children were all below the 30th percentile for the body mass index. The whole-body protocols showed similar increases in bone mineral density with age; however, the experimental paediatric protocol always produced a lower bone mineral density result than the adult protocol. Paired results showed limits of agreement of 0.0668-0.130 g x cm(-2) (male, 0.063-0.124 g x cm(-2); female, 0.073-0.134 g x cm(-2)). This represents a mean difference of 11%. Five results showed differences greater than the upper limit of agreement. CONCLUSIONS: For anteroposterior (AP) lumbar spine scans, the use of the paediatric algorithm in children under 11 years of age would prevent the largest failures in analysis. For whole-body scanning, the adult algorithm showed no major failures in children of 11 years or older. It is hoped that forthcoming improvements in whole-body density analysis will improve the results for those under 11 years of age. Normal range data should be generated for any new algorithm to allow proper interpretation of clinical studies.