Aerosolised hyaluronic acid prevents exercise-induced bronchoconstriction, suggesting novel hypotheses on the correction of matrix defects in asthma
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.
Section snippets
Production plan for the preparation of the HA used in the present study
Preliminarly, solutions of HA with a molecular weight variable from 400 to 4000 kilodaltons (KD) were prepared [7]. A condensation reaction between glucuronic acid of vegetal origin and hydrolysed chitin solution (acetylglucosamine 85–90% the dry weight) in presence of a biological catalysator, at a temperature of 18–20 °C, is successively obtained. A 10% water mixture of about 90% disaccharide (provided with a structure which is identical to the HA basic monomer) and HA polymers for the resting
Results
The bronchoconstrictive effect induced by physical exercise has been relevant (Fig. 3) and statistically significant (Table 1): the average FEV1 measured 5 min post-exercise was reduced by 36.14% from the baseline values (p<0.0001) which have been obtained in the subjects pre-treated with aerosolised P.
Pre-treatment with aerosolised HA determined partial but clear protection on the FEV1 reduction due to the bronchoconstriction induced by the challenge: the average post-challenge FEV1 was proven
Discussion
The physiological and physico-chemical HA properties derive from its molecular structure which in solution is a casual winding. This occupies a volume which is 1000 times larger than that occupied by organic materials [34], so that the neighbouring molecules are trapped by HA in concentrations >1 mg/ml [35].
Since two molecules cannot occupy the same space in the same time and since the volume occupied by HA is very large, HA solutions of 5 or 15 mg/ml exclude albumin, respectively, from 25 to 75%
Acknowledgements
The authors acknowledge Valter Fasano, Antonio Comi and Lisa Longo for technical assistance and particularly appreciate Sabrina Della Patrona for assistance with data management and analysis.
References (42)
- et al.
Molecular mechanisms and genetics of hyaluronan biosynthesis
Intern J Biol Macromol
(1994) - et al.
Turnover of hyaluronan in the tissues
Adv Drug Deliv Rev
(1991) Hyaluronan-binding proteins and receptors
Adv Drug Deliv Rev
(1991)- et al.
Density-dependent expression of hyaluronic acid binding to vascular cells in vitro
Microvasc Res
(1991) - et al.
Glycosaminoglycans, airways inflammation and bronchial hyperresponsiveness
Pulm Pharmacol Ther
(2001) - et al.
On the interaction between polysaccharides and other macromolecules
Biochem Biophys Acta
(1963) - et al.
Turnover of hyaluronan in the microcirculation
Am Rev Respir Dis
(1992) - et al.
Hyaluronan in the rat with special references to the skin
Acta Physiol Scand
(1988) - et al.
Uptake and degradation of hyaluronan in lymphatic tissue
Biochem J
(1988) - et al.
Scavenger function of the liver endothelial cell
Biochem J
(1990)
Effect of colture conditions on rates of intrinsic hyaluronic acid production by Streptococcus equi spp. Zoepidermicus
Biotechnol Lett
Hyaluronate is synthetized at plasma membranes
Biochem J
Hyaluronic acid and erythrocyte flexibility
Intern J Microcirc Clin Exp
An overview of proteoglycans in physiology and pathology
Hyaluronan affects extravascular water in lung of anesthetized rabbits
J Appl Physiol
Glycosaminoglycans and morphogenesis
Inhibition of leukocyte locomotion by hyaluronic acid
J Cell Sci
Modulation of chemotaxis and migration of PMN cells by hyaluronic acid
Z Rheumatol
Effect of hyaluronic acid on neutrophil adhesion
J Cell Sci
The role of hyaluronic acid in arthritis and its therapeutic use
Modification of chemiotaxis by sinovial fluid hyaluronate
Arthritis Rheum
Cited by (69)
Performance and biocompatibility of a novel inhalable dry powder formulation based on hyaluronic acid intended to protect the respiratory tract mucosa
2023, International Journal of PharmaceuticsHyaluronan in the pathogenesis of acute and post-acute COVID-19 infection
2023, Matrix BiologyThe effects of female sexual hormones on the endothelial glycocalyx
2023, Current Topics in MembranesModulation of hyaluronan signaling as a therapeutic target in human disease
2022, Pharmacology and TherapeuticsCo-Spray-Dried Urea Cross-Linked Hyaluronic Acid and Sodium Ascorbyl Phosphate as Novel Inhalable Dry Powder Formulation
2019, Journal of Pharmaceutical SciencesIn vitro characterization of physico-chemical properties, cytotoxicity, bioactivity of urea-crosslinked hyaluronic acid and sodium ascorbyl phosphate nasal powder formulation
2019, International Journal of PharmaceuticsCitation Excerpt :High-molecular weight (≥1 MDa) hyaluronic acid (HA) is an important component of the normal airway secretions, produced by submucosal glands and by superficial airway epithelial cells (Basbaum and Finkbeiner, 1988; Fallacara et al., 2018a; Monzon et al., 2006). HA plays a central role in the homeostasis of the whole respiratory apparatus, influencing bio-mechanical forces, hydric balance, cellular functions, growth factors’ activity and cytokines’ behaviour (Casale et al., 2016; Fallacara et al., 2018a; Petrigni and Allegra, 2006). In the nasal epithelium, HA promotes mucociliary clearance and sustains mucosal surface healing (Gelardi et al., 2013a).