Aerosolised hyaluronic acid prevents exercise-induced bronchoconstriction, suggesting novel hypotheses on the correction of matrix defects in asthma

Abstract

Hyaluronic acid (HA), a biopolymer, member of the family of the glycosaminoglicanes (GAGs) is one of the major natural components of the connective amorphous matrix. The lungs, together with skin and intestine, contain >50% of HA of the body: it provides to several biologic functions and presents the unique capacity to link and retain a particularly relevant number of water molecules. Since other GAGs have been proven to be provided with anti-asthmatic properties and HA has been employed with positive results by intra-tracheal instillation in experimental models of lung emphysema and COPD, we have explored the efficacy of the pre-administered aerosol of HA (compared to placebo) in preventing in asthmatic patients the bronchoconstriction induced by a challenge test such as that obtained with muscular exercise. In a randomised, cross-over, single-blind study design, saline as placebo (P) or HA have been administered by aerosol, in two non-consecutive days, 30 min prior to the beginning of the challenge (10 min free running), to 14 patients (13–36 years old; 7 teenagers, 7 young adults; 11 males, 3 females; 12 allergic, 2 non-allergic), all suffering from mild bronchial asthma. The bronchoconstrictive effect induced by the muscular exercise has been relevant and statistically significant. With the P pre-treatment, the average FEV1 measured 5 min after the end of exercise was reduced by 36.14% from the baseline FEV1. Pre-treatment with HA determined a partial but clear-cut protection of the FEV1 impairment due to the challenge: the average post-challenge FEV1 resulted to be 12.43% less than the pre-challenge baseline value. No significant difference was observed in the level of HA protection in the subgroup of teenagers when compared to that of young adults. The protection induced by HA, when compared with P, resulted particularly significant by the statistical point of view (p<0.0001). We conclude that aerosol HA administration significantly reduces the bronchial hyper-reactivity to muscular exercise in asthmatics. Such effect could be attributed to the correction of the pathological remodelling, one of the main features of asthma: a correction which could be attributed to the unique physicochemical properties of this major component of the loose connective amorphous matrix of the airways, which is undoubtedly involved in the remodelling process.

Introduction

Hyaluronic acid (HA) is a naturally occurring biopolymer which serves several important biological functions in bacteria and superior organisms, including human. In human tissues, naturally occurring HA may be found ubiquitously, in particular as intercellular space filler of the extra-cellular matrix, with higher concentration in the vitreous humour and synovial liquid, but also in the loose connective tissue [1].

HA is a member of the family of the glycosaminoglicanes (GAGs): the molecule is constituted by a linear chain without ramifications, containing a repeated series of disaccharide units of glucuronic acid and N-acetyl-glucosamine linked by glucoside chains (Fig. 1). One single molecule contains up to 10,000 disaccharide units and it may have a molecular weight of several million Daltons. Different from other GAGs, HA does not contain sulphur groups, is free from covalent links to proteins and presents the simplest chemical structure.

HA presents the unique capacity to link and retain a relevant number of water molecules in the inter-fibrillar spaces contributing to constitute the fundamental part of the amorphous colloidal matrix which glues cells and connective fibres, and to determine relevant effects on tissue morphogenesis [2], cells growth, differentiation and migration, hydration, lubrication, solute transport, as well as on microcirculatory exchanges, due to its influence on interstitial volume, hydraulic conductibility and macromolecule diffusibility [3].

In serum, HA levels are 10–100 μg/l. Skin, intestine and lung contain more than 50% of HA of the body [4]. In the lungs, HA content is 15–150 μg/g dry weight (different species), mainly localised in the peri-bronchial and inter-alveolar/peri-alveolar tissue. It is drained by the lymphatic vessels and catabolised in the local lymph nodes [5] and liver [6].

Up to some years ago, HA was extracted from natural sources such as cockscomb or bovine connective tissues and the procedure consisted in enzymatic digestion and specific separation from the proteins in order to purify the extract. This procedure presented several disadvantages: bad molecular weight standardisation, risk of viral infection, high costs.

More recently [7], such disadvantages have been largely avoided and good results have been obtained thanks to a biotechnological procedure consisting in the bio-fermentation of G+ bacteria such as Streptococcus zooepidermicus (Fig. 2), a micro-organism which is coated with a mucoid HA capsule and can so theoretically provide unlimited HA biopolymers with a molecular weight going from 10,000 up to 4 million Daltons (depending on temperature, initial glucose concentrations, speed of bacterial growth): this is presently the favourite production procedure worldwide.

HA is synthesised into the fibroblast and other cellular plasmatic membranes through the joining of glycidic residuals to the reducing chain extremity [8]. HA receptors have been localised on fibroblasts and 3T3 cells surface [9] and recently on endothelial surface [10] probably influencing angiogenesis. Furthermore, HA reduces erythrocite viscosity [11].

Although less investigated than heparin, HA, due to its peculiar physico-chemical characteristics, has been proven to be provided with several biologic functions in the tissues of animals and humans, such as stabilisation of proteoglicans in the extra-cellular matrix [12], high hydrating properties [13], contribution to tissue repair [14], inhibition of migration, chemiotaxis and aggregation of polymorphonuclear leucocytes and monocytes [15], [16], [17], regulation of cellular growth and solute transfer.

Furthermore, its concentration is high in several tissues and parenchymas, large quantity of HA being especially present in the lung. Therapeutic applications of HA in diseases interesting domains such as the orthopaedic, ophthalmic and dermatologic ones have already been extensively studied and reproduced in animal models [18], [19], [20], [21], [22].

Together with its molecular similarities with other GAGs, especially heparin [23] which according to the literature is provided with anti-asthmatic properties [24], [25], [26], [27], [28], the above mentioned HA biological activities and characteristics give evidence in favour of its possible therapeutic interventions in such respiratory disease. Some studies have been performed on the activity of HA in models of other obstructive diseases such as emphysema and exacerbation of COPD [29], [30]; our preliminary data have shown the possibility of preventing induced bronchoconstriction in asthma thanks to the pre-treatment with aerosolised HA [31], [32]: such aerosol had been proven to protect the asthmatics from different specific and non-specific challenges [33]. In this study we present data on the effect of inhaled HA in adult asthmatics, namely on the bronchial response to a non-specific challenge such as physical exercise.