High Molecular Weight Hyaluronan Reverses Acid Instillation Induced Acute Lung Injury

Part I HCl instillation induces lung injury and inflammation as well as increases L-HA in BALF and lung tissueObjective Acid(HCl)aspiration during anesthesia may lead to acute lung injury.There is no effective therapy.This study aims to establish HCl instillation induced murine acute lung injury model and identify the change of low molecular weight hyaluronan(L-HA)in vivo.Method We intratracheal instilled HCl(0.1 M,p H 1.25,2 ml/kg)in C57BL/6 wild type(WT)mice.Bronchoalveolar lavage fluid(BALF)were collected at 1,5 or 24 h post HCl and protein concentration,total and differential cell count,HA and its binding partner protein IαI levels were measured.Agarose gel images were used to detect different size of HA and FITClabeled hyaluronic acid-bind proteins(HABPs)were detected in paraformaldehyde-fixed murine lung tissue.Our study also collected BALF samples from health volunteers and mechanically-ventilated patients to measure the concentration and sizes of HA.Results When instilled intratracheally into C57BL/6 mice,HCl resulted in significant increased level of protein in BALF 24 hours later,as well as induced an increased number of inflammatory cells starting at 1 hour and persisting at 24 hours post exposure compared with mice received normal saline(NS).At 24 hours post HCl instillation there were significant increases of HA in BALF compared with mice received NS.Moreover,elevated levels of IαI were seen at as early as 1 hour after HCl instillation and persisted at 24 hours post exposure.Furthermore,the levels of HABP expression in HCl instilled lung tissue increased gradually compared with those from mice instilled with NS.Agarose gel images from HCl instilled mice,showed the presence of L-HA fragments at 5 and 24 hours post HCl exposure,the intensity of which increased at 5 and 24 hours after HCl instillation.We could not detect any HA in BALF of normal healthy volunteers.However,HA levels in BALF are significantly elevated in mechanically-ventilated patients compared with healthy volunteers.There was a continuous smear from higher than 1,510 k Da to below 30 k Da,suggesting that HA might be accounting for the observed staining.Conclusion HCl instillation could induce inflammation and lung injury as well as increases L-HA in BALF and lung tissuePart II Instillation of Hyaluronan Reverses Acid Instillation Injury to the Mammalian Blood Gas BarrierObjective We have identified that HCl instilled intratracheally in C57BL/6 mice results in the formation of L-HA,then we hypothesized that L-HA activates Rho A and ROCK,causing airway hyperresponsiveness(AHR)and increased permeability.Instillation of high molecular weight hyaluronan(H-HA),reverses lung injury.Method We instilled HCl(0.1 M,p H 1.25,2 ml/kg)in C57BL/6 wild type(WT),MPO(-/-)and CD44(-/-)mice.WT mice were also instilled intranasally with high molecular weight hyaluronan(H-HA)at 1 and 23 h post HCl.All measurements were performed at 1,5 or 24 h post HCl.The effects of H-HA to acute lung injury were observed.Lung tissue slices were stained with HE or MPO to determine the severity of lung injury and infiltration of neutrophils.Mice BLAF were collected to measure protein concentration,total and differential cell count,HA level and sizes.Mice Respiratory mechanics,pulmonary filtration coefficient(Kf)and wet-to-dry ratio were also measured.Expression of lung Rho A and its downstream kinase ROCKs were detected by immunoblotting.Expression of hyaluronan synthases(HAS)were detected by quantitative Real-Time PCR.Primary human bronchial epithelial cell barrier integrity was measured using an Electric Cell-substrate Impedance Sensing system.Results Instillation of HCl in WT but not in CD44(-/-)resulted in increased inflammation,AHR,lung injury and L-HA in the BALF 24 hours post HCl;L-HA levels and lung injury were significantly lower in HCl-instilled MPO(-/-)mice.Isolated perfused lungs of HCl instilled WT but not of CD44(-/-)mice had elevated values of the Kf.Addition of L-HA on the apical surface of human primary bronchial epithelial cell monolayer,decreased barrier resistance(RT).H-HA significantly mitigated inflammation,AHR and pulmonary vascular leakage at 24 h after HCl instillation and mitigated the increase of Kf and RT,as well as ROCK2 phosphorylation and HAS1 and HAS2.Conclusion HCl instillation-induced lung injury is mediated by the L-HA-CD44-Rho AROCK2 signaling pathway and H-HA is a novel therapeutic agent for acute lung injury.