Another Step forward in Cystic Fibrosis

It was interesting to read the recent paper in the ISME journal by Whiteson and colleagues (1) who showed that Cystic Fibrosis (CF) patients that harbour high levels of the bacteria P. Aeruginosa and R. Mucilaginosa in their airways also produce higher levels of 2,3-butanedione (diacetyl), an agent that is used extensively in popcorn flavouring and which has shown to damage airway epithelium in rats (2).

CF is an autosomal recessive condition that affects approximately in 3,000 new births. The pathophysiology involves accumulation of thick mucus in the airways which can lead to progressive lung damage and is also associated with impaired digestion and infertility. The hypoxic environment of mucus-clogged airways provides an ideal niche for the aforementioned microorganisms. The lung damage  is thought to occur mainly during periods of exacerbation following periods of relative stability, which can be restored by intravenous antibiotic administration. The mechanisms via which the microorganisms may cause lung damage are relatively poorly understood but this paper provides a major step forward.

Using analysis of breath gas from CF patients and non-CF volunteers the authors found diacetyl to be present in both groups but with elevation in some CF patients. Metagenome sequencing of the most common bacterial populations in CF sputum revealed genes involved in acetoin metabolism were more common in streptococcus species and the authors postulate that diacetyl produced by streptococci during metabolic adaptation to hypoxia and low pH may drive the production of phenazine from other microbial species, such as P. Aeruginosa. Phenazines have been shown in other studies to induce expression of chemokines and adhesion molecules in airway epithelia that lead ultimately to enhanced immune cell infiltration and protracted inflammation (3). Recent data also show phenazine concentrations correlate negatively with lung function (4).

Clearly the signalling cross-talk between micro-organisms, epithelial cells and immune cells is complex and altered microbial metabolism is likely to be one aspect of a multifaceted pathogenic mechanism. This study provides a starting point which requires validation with larger samples. Identifying imminent exacerbations in CF patients is currently not possible so the discovery of novel biomarkers, of which increased diacetyl production may be one, is needed to provide rapid treatment and ultimately improve survival.

References

1.  Whiteson et al. Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation ISME Journal 2014 to be found here

2.  Zaccone et al. Popcorn Flavouring Effects on Reactivity of Rat Airway in vivo and in vitro Journal of Toxicology and Environmental Health  2013

3. Look et al. Pyocanin and Its Precursor Phenazine-1-Carboxylic Acid Increase IL-8 and Intercellular Adhesion Molecule-1 Expression in Human Airway Epithelial Cells by Oxidant-Dependent Mechanisms Journal of Immunology 2005

4. Hunter et al. Phenazine Content in The Cystic Fibrosis Respiratory Tract Negatively Correlates with Lung Function and Microbial Complexity Respiratory Cell and Molecular Biology 2012

The anti-angiogenic potential of targeting Prostaglandin E2 receptors in tumours

Tumour hypoxia refers to the gradual decrease in partial pressure of oxygen as tumours grow. Cells more than 1-2 cm distant from blood vessels therefore have reduced access to oxygen and nutrients. Tumour cells, however, display a coordinated response to hypoxia and release a number of factors which are involved in processes such as metabolic reprogramming and angiogenesis which promote their growth further and provide a route for metastasis.

Angiogenesis can broadly be defined as the growth of blood vessels from an existing vascular bed, which is dependent on endothelial cell proliferation, migration and tube formation. In healthy individuals endothelial cells are normally quiescent and only undergo proliferation in a highly regulated manner during processes such as wound healing and repair of the uterine lining. This quiescence is due to the presence of a broad group of endogenous anti-angiogenic factors which override pro-angiogenic stimuli. In tumours, however, the balance is shifted in favour of pro-angiogenic factors which mobilise endothelial cells; a phenomenon referred to as the 'angiogenic switch'.

Tumour cells are known to release a number pro-angiogenic factors during hypoxia, the best characterised of which is VEGF. There is considerable evidence, however, that prostaglandin E2 (PGE2) is also upregulated during hypoxia in a number of solid tumours, and this in itself is a potent angiogenic factor. PGE2 is synthesised downstream of COX-2, an enzyme which is well recognised therapeutic target for inflammation. While COX-2 inhibition has displayed success in preventing tumour growth, its prolonged inhibition in vivo is associated with a number of complications that are discussed in more detail below.


The mechanisms via which PGE2 promotes angiogenesis are not well understood. This is an area I will be researching from April to August as part of my Master's dissertation. In the meantime, I am conducting a literature review to consolidate the current understanding of PGE2 in angiogenesis and the tumour microenvironment.

PGE2 binds to a series of cognate G-protein coupled receptors termed EP1-4. These receptors are coupled to diverse intracellular signalling cascades and display heterogeneous expression across different tissues. While endothelial cells express all four receptors, the expression pattern on other cells within the tumour microenvironment, including cancer associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs) and tumour associated macrophages (TAMs) remains to be elucidated. It is known, however, that  tumour-derived PGE2 can promote angiogenesis directly through endothelial cells, but can also influence stromal cells which themselves release pro-angiogenic molecules. I have attempted to clarify this complex relationship in the diagram below:

It is clear PGE2 is a key player at a number of steps. It can polarize TAMs to an M2 phenotype to promote inflammation and release pro-angiogenic factors; it is involved in induction of MDSCs, which secrete a number of immunosuppressive and pro-angiogenic factors and can also act on CAFs. Moreover, PGE2 and VEGF may promote endothelial cell mobilisation via an autocrine mechanism.

Non-steroidal antiflammatory drugs (NSAIDs) have long been recognised to attenuate inflammation and reduce cancer risk through COX-1 and COX-2 inhibition. Prolonged use of NSAIDs is however associated with adverse events, most common of which are gastric ulcers, owing to the important role of COX-1- derived prostanoids in homeostasis across different tissues. The advent of COX-2 specific inhibitors such as celecoxib attempted to rectify this problem. While initially successful, a number of longitudinal studies have linked high long-term doses of these compounds to adverse cardiovascular events. This may reflect reduced biosynthesis of COX-2 derived anti-thrombotic PGI2 with sustained production of COX-1-derived pro-thrombotic Thromboxane A2. More specific compounds which modulate levels of individual prostanoids are clearly needed.

The focus of my research over the four months will be to understand which EP receptors are important to angiogenesis and the intracellular cascades mediating their effects. Targeting the terminal prostaglandin E2 synthase enzymes  may be a viable strategy to specifically impair angiogenesis while leaving levels of other prostanoids unaltered. Similarly, specific targeting of EP receptors on tumour and stromal cells may one day prove useful as monotherapy or in conjunction with current anti-angiogenic therapies such as bevacizumab.

Oxidised ApoA1- A major step in atherosclerosis and CVD

A recent study by Huang and colleagues published in Nature Medicine has helped to elucidate the mechanism whereby 'good cholesterol' high density lipoproteins (HDLs) lose their cardioprotective function and contribute to inflammation, especially associated with risk of atherosclerosis (1). More than 75% of HDL is composed of apolipoprotein A1 (ApoA1) and it is this component that is essential for the beneficial effects of HDLs. Briefly, native HDLs have been shown to reduce inflammation, target cholesterol for degradation, increase cellular insulin sensitivity and inhibit coagulation (Gordts et al. 2014)(2). Understanding HDL loss-of- function in vivo may therefore assist the quest for therapeutic targets that maintain native cardioprotective HDL. Previous studies from CVD patients have shown ApoA1 in the artery wall to be oxidatively cross-linked and dissociated from HDLs. Myeloperoxidase (MPO), mainly derived from macrophages, is the major enzyme responsible for these modifications.

This study was the first to examine which site-specific modification of ApoA1 was responsible for its dysfunction. It was found that oxidation of tryptophan at codon 72  (Trp72) was oxidised by MPO, and this form recovered from patients was found to be lipid-poor and capable of inducing expression of inflammatory genes in endothelial cells, the first step to atherosclerosis. In addition, oxTrp-72 ApoA1 showed reduced cholesterol binding, thereby elevating circulating cholesterol levels. One of the most significant findings to emerge is that the oxTrp72 ApoA1 to native ApoA1 ratio in cardiology patients was a strong predictor of subsequent CVD and atherosclerosis incidence.

This study raises the possibility for using MPO as a therapeutic target while levels of oxTrp72 ApoA1 may be a useful prognostic indicator. Ronald et al. have already exploited MRI scanning that detects MPO activity in vivo to image areas of atherosclerotic inflammation noninvasively(3). Clearly, the mechanisms by which mediate HDL cardioprotection will continue to come to light, helping to treat an increasingly prevalent disease in the Western world.

References

1. Huang et al. An abundant dysfunctional apolipoprotein A1 in human atheroma Nature Medicine January 2014
2.  Gordts et al. Pleiotropic effects of HDL: Towards new therapeutic areas for HDL-targeted interventions Current Molecular Medicine  November 2013
3. Ronald et al. Enzyme-sensitive magnetic resonance imaging targeting myeloperoxidase identifies active inflammation in experimental rabbit atherosclerotic plaques. Circulation August 2009