Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation.

The regulatory function of plasma-membrane Ca(2+)-ATPase (PMCA) in the heart.

The PMCA (plasma-membrane Ca(2+)-ATPase) is a ubiquitously expressed calcium-extruding enzymatic pump important in the control of intracellular calcium concentration. Unlike in non-excitable cells, where PMCA is the only system for calcium extrusion, in excitable cells, such as cardiomyocytes, PMCA has been shown to play only a minor role in calcium homoeostasis compared with the NCX (sodium/calcium exchanger), another system of calcium extrusion. However, increasing evidence points to an important role for PMCA in signal transduction; of particular interest in cardiac physiology is the modulation of nNOS (neuronal nitric oxide synthase) by isoform 4b of PMCA. In the present paper, we will discuss recent advances that support a key role for PMCA4 in modulating the nitric oxide signalling pathway in the heart.

GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon.

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, deltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551) K18-GFP-CFTR(+/-), designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression.

The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.

G551D CF mice display an abnormal host response and have impaired clearance of Pseudomonas lung disease.

Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-alpha, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.