Hari G. Garg

Regulation of lung injury and repair by Toll-like receptors and hyaluronan

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88−/− and Tlr4−/−Tlr2−/− mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell–specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell–surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-κB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.

Hyaluronan and wound healing: a new perspective

Hyaluronan has long been associated with the remodelling extracellular matrix. Such remodelling occurs in development, growth and wound healing. This role has been thought to be related to the physical structure and chemical composition of the pure glycosaminoglycan chain. We question this proposition and present evidence which suggests that proteins associated with hyaluronan may be more critical determinants of tissue remodelling.

Cyclin-Dependent Kinase Inhibitor p27Kip1, But Not p21WAF1/Cip1, Is Required for Inhibition of Hypoxia-Induced Pulmonary Hypertension and Remodeling by Heparin in Mice

Heparin has growth inhibitory effects on pulmonary artery smooth muscle cell (PASMC) in vitro and in vivo. However, the mechanism has not been fully defined. In this study, we investigated the role of cyclin-dependent kinase inhibitors, p21WAF1/cip1 (p21) and p27Kip1 (p27), in the inhibitory effect of heparin on PASMC proliferation in vitro and on hypoxia-induced pulmonary hypertension in vivo using p21 and p27-null mice. In vitro, loss of the p27 gene negated the inhibitory effect of heparin on PASMC proliferation, but p21 was not critical for this inhibition. In vivo, heparin significantly inhibited the development of hypoxia-induced pulmonary hypertension and remodeling, as evidenced by decreased right ventricular systolic pressure, ratio of right ventricular weight to left ventricle plus septum weight, and percent wall thickness of pulmonary artery, in p21+/+, p21−/−, p27+/+, and p27+/−, but not in p27−/− mice. We also observed that hypoxia decreased p27 expression significantly in mouse lung, which was restored by heparin. Heparin inhibited Ki67 proliferative index in terminal bronchial vessel walls in p27+/+ and p27+/−, but not in p27−/− mice exposed to hypoxia. Therefore, we conclude that the cyclin-dependent kinase inhibitor p27, but not p21, is required for the inhibition of hypoxic pulmonary vascular remodeling by heparin.

High-molecular-weight hyaluronan – a possible new treatment for sepsis-induced lung injury: a preclinical study in mechanically ventilated rats

IntroductionMechanical ventilation with even moderate-sized tidal volumes synergistically increases lung injury in sepsis and has been associated with proinflammatory low-molecular-weight hyaluronan production. High-molecular-weight hyaluronan (HMW HA), in contrast, has been found to be anti-inflammatory. We hypothesized that HMW HA would inhibit lung injury associated with sepsis and mechanical ventilation.MethodsSprague–Dawley rats were randomly divided into four groups: nonventilated control rats; mechanical ventilation plus lipopolysaccharide (LPS) infusion as a model of sepsis; mechanical ventilation plus LPS with HMW HA (1,600 kDa) pretreatment; and mechanical ventilation plus LPS with low-molecular-weight hyaluronan (35 kDa) pretreatment. Rats were mechanically ventilated with low (7 ml/kg) tidal volumes. LPS (1 or 3 mg/kg) or normal saline was infused 1 hour prior to mechanical ventilation. Animals received HMW HA or low-molecular-weight hyaluronan via the intraperitoneal route 18 hours prior to the study or received HMW HA (0.025%, 0.05% or 0.1%) intravenously 1 hour after injection of LPS. After 4 hours of ventilation, animals were sacrificed and the lung neutrophil and monocyte infiltration, the cytokine production, and the lung pathology score were measured.ResultsLPS induced lung neutrophil infiltration, macrophage inflammatory protein-2 and TNFα mRNA and protein, which were decreased in the presence of both 1,600 kDa and 35 kDa hyaluronan pretreatment. Only 1,600 kDa hyaluronan completely blocked both monocyte and neutrophil infiltration and decreased the lung injury. When infused intravenously 1 hour after LPS, 1,600 kDa hyaluronan inhibited lung neutrophil infiltration, macrophage inflammatory protein-2 mRNA expression and lung injury in a dose-dependent manner. The beneficial effects of hyaluronan were partially dependent on the positive charge of the compound.ConclusionsHMW HA may prove to be an effective treatment strategy for sepsis-induced lung injury with mechanical ventilation.

Deficiency of the NHE1 gene prevents hypoxia-induced pulmonary hypertension and vascular remodeling.

RATIONALE Our previous studies found that Na(+)/H(+) exchanger (NHE) activity played an essential role in pulmonary artery smooth muscle cell (PASMC) proliferation and in the development of hypoxia-induced pulmonary hypertension and vascular remodeling. Other investigators recently observed increased expression of the NHE isoform 1 (NHE1) gene in rodents with pulmonary hypertension induced by hypoxia. However, a causal role for the NHE1 gene in pulmonary hypertension has not been determined. OBJECTIVES To determine the causal role of the NHE1 gene in pulmonary hypertension and vascular remodeling. METHODS We used NHE1-null mice to define the role of the NHE1 gene in the development of pulmonary hypertension and remodeling induced by hypoxia and to delineate the NHE1 regulatory pathway. MEASUREMENTS AND MAIN RESULTS After 2 weeks of exposure to hypoxia, in contrast to wild-type hypoxic littermates, there was no significant increase in right ventricular systolic pressure, in the ratio of right ventricular to left ventricular plus septal weight [RV/(LV + S)], or in medial wall thickness of the pulmonary arterioles in homozygous mice (NHE1(-/-)). There was a significant decrease in Rho kinase (ROCK1 and ROCK2) expression, accompanied by an increase in p27 expression in NHE1(-/-) mice. CONCLUSIONS Our study demonstrated that deficiency of the NHE1 gene prevented the development of hypoxia-induced pulmonary hypertension and vascular remodeling in mice and revealed a novel regulatory pathway associated with NHE1 signaling.

Effect of proteoglycans on type I collagen fibre formation.

Collagen fibrillogenesis is a multistep process involving assembly of molecules into fibrils and bundles of fibrils. The exact role of proteoglycans in collagen fibrillogenesis is unclear. The purpose of these studies is to study the effect of proteoglycans on collagen fibrillogenesis in vitro. Results of these studies suggest that the proteoglycans dermatan sulphate and chondroitin sulphate do not change the final turbidity and hence the diameter of fibrils formed during the early stages of fibrillogenesis. This suggests that proteoglycans may not influence the early phases of collagen assembly, such as nucleation. However, proteoglycans added during the final stages of collagen fibre formation in vitro cause changes in ultimate tensile strength. In the presence of the high-molecular-weight proteoglycan, the ultimate tensile strength is increased by a factor of 1.5 above that of the control, whilst in the presence of low-molecular weight chondroitin sulphate proteoglycan the tensile strength is significantly decreased. It is concluded that proteoglycans influence the later stages of fibre formation. The presence of high-molecular-weight chondroitin sulphate proteoglycan leads to efficient stress transfer between collagen fibrils, altering the ultimate tensile strength. The results of these studies will be useful in optimizing the design of collagen tendon-ligament prostheses.

TLR4 Is a Negative Regulator in Noninfectious Lung Inflammation

Low m.w. hyaluronan (LMW HA) has been shown to elicit the expression of proinflammatory cytokines and chemokines in various cells in vitro. However, the effects of this molecule in vivo are unknown. In this study, we report that intratracheal administration of LMW HA (200 kDa) causes inflammation in mouse lung. A lack of TLR4 is associated with even stronger inflammatory response in the lung as shown by increased neutrophil counts and elevated cytokine and chemokine concentrations. We also demonstrate that TLR4 anti-inflammatory signaling is dependent upon a MyD88-independent pathway. TLR4-mediated IL-1R antagonist production plays a negative regulatory role in LMW HA (200 kDa) induced lung inflammation. These data provide a molecular level explanation for the function of TLR4 in LMW HA (200 kDa)-induced lung inflammation, as inhibition of the β form of pro–IL-1 promotes an anti-inflammatory response.

Human Skin and Post-Burn Scar Hyaluronan: Demonstration of the Association with Collagen and other Proteins

Hyaluronan (HA) extracted from tissues has been demonstrated to have an enhancing effect on the process of wound healing; the question arises whether this effect is due to the HA or to associated collagen and other proteins. In this study, HA has been extracted from human skin and scar tissue under dissociative conditions and isolated by DEAE-cellulose chromatography followed by CsCl gradient and Sepharose CL-6B chromatography. This highly purified HA was found to contain between 4 and 28% protein, with collagen constituting 5% of the total protein. A functional association between HA and a collagen protein complex is proposed.

High MW hyaluronan inhibits smoke inhalation-induced lung injury and improves survival

Background and objective:  High MW hyaluronan (HMW HA) as opposed to low MW hyaluronan (LMW HA) has been shown to have anti‐inflammatory and anti‐apoptotic effects. We hypothesized that treatment with HMW HA would block smoke inhalation lung injury by inhibiting smoke‐induced lung inflammation and airway epithelial cell apoptosis.

Synthese eines xylose-haltigen glycopeptides, das die aminosäure-sequenz 4 bis 7 des NH2-terminus der protein-core-struktur des rinderhaut-proteodermatan-sulfats enthält☆

Abstract The synthesis is described of α-β- d -xylopyranosyl- l -serylglycyl- l -isoleucylglycine (7), a glycopeptide containing the amino acid sequence of the protein core-structure of beef-skin proteodermatan sulfate; coupling of N-protected O-XylpSer with protected GlyIleuGly followed by deprotection afforded 7.