Poloxamer 188 facilitates the repair of alveolus resident cells in ventilator-injured lungs.

RATIONALE: Wounded alveolus resident cells are identified in human and experimental acute respiratory distress syndrome models. Poloxamer 188 (P188) is an amphiphilic macromolecule shown to have plasma membrane-sealing properties in various cell types. OBJECTIVES: To investigate whether P188 (1) protects alveolus resident cells from necrosis and (2) is associated with reduced ventilator-induced lung injury in live rats, isolated perfused rat lungs, and scratch and stretch-wounded alveolar epithelial cells. METHODS: Seventy-four live rats and 18 isolated perfused rat lungs were ventilated with injurious or protective strategies while infused with P188 or control solution. Alveolar epithelial cell monolayers were subjected to scratch or stretch wounding in the presence or absence of P188. MEASUREMENTS AND MAIN RESULTS: P188 was associated with fewer mortally wounded alveolar cells in live rats and isolated perfused lungs. In vitro, P188 reduced the number of injured and necrotic cells, suggesting that P188 promotes cell repair and renders plasma membranes more resilient to deforming stress. The enhanced cell survival was accompanied by improvement in conventional measures of lung injury (peak airway pressure, wet-to-dry weight ratio) only in the ex vivo-perfused lung preparation and not in the live animal model. CONCLUSIONS: P188 facilitates plasma membrane repair in alveolus resident cells, but has no salutary effects on lung mechanics or vascular barrier properties in live animals. This discordance may have pathophysiological significance for the interdependence of different injury mechanisms and therapeutic implications regarding the benefits of prolonging the life of stress-activated cells.

Determinants of plasma membrane wounding by deforming stress.

Once excess liquid gains access to air spaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that interventions such as a hypertonic treatment increase adhesive PM-cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells and fibroblasts and measured their retractive force as readout of PM-CSK adhesive interactions using optical tweezers. A 50-mOsm increase in media osmolarity consistently increased the tether retractive force in epithelial cells but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with latrunculin, cytochalasin D, and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel, and the fraction of wounded cells were measured. Interventions that increased PM-CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleural alveolus resident cells to baseline levels. Our observations support the hypothesis that PM-CSK adhesive interactions are important determinants of the cellular response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury.

Impact of buffering hypercapnic acidosis on cell wounding in ventilator-injured rat lungs.

We measured the effects of raising perfusate pH on ventilator-induced cell wounding and repair in ex vivo mechanically ventilated hypercapnic rat lungs. Lungs were randomized to one of three perfusate groups: 1) unbuffered hypercapnic acidosis, 2) bicarbonate-buffered hypercapnia, or 3) tris-hydroxymethyl aminomethane (THAM)-buffered hypercapnia. The membrane-impermeant label propidium iodide was added to the perfusate either during or after injurious ventilation providing a means to subsequently identify transiently wounded and permanently wounded cells in optical sections of subpleural alveoli. Normalizing perfusate pH in hypercapnic preparations attenuated ventilator-induced cell injury, particularly in THAM-buffered preparations. This was observed despite greater amounts of edema and impaired lung mechanics compared with other treatment groups. Protective effects of buffering of hypercapnic acidosis on injury and repair were subsequently confirmed in a cell scratch model. We conclude that buffering of hypercapnic acidosis attenuates plasma cell injury induced by mechanical hyperinflation.

Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation.

Treatment of acute cellular rejection (ACR) is associated with increased viral load, more severe histologic recurrence, and diminished patient and graft survival after liver transplantation for hepatitis C virus (HCV). Recurrence of HCV may be difficult to distinguish histologically from ACR. Because the immunologic mechanisms of ACR and HCV recurrence are likely to differ, we hypothesized that ACR is associated with the expression of a specific subset of immune activation genes that may serve as a diagnostic indicator of ACR and provide mechanistic insight into the pathophysiology of ACR and recurrence of HCV. The goal of the study was to study intragraft gene expression patterns in ACR and during recurrence of HCV in HCV-infected recipients. High-density microarrays were used to determine relative intragraft gene expression in two groups of HCV-infected liver transplant recipients: four with steroid responsive ACR by Banff criteria and four age- and gender-matched HCV-infected recipients with similar necroinflammatory activity but without histological criteria for rejection (no cholangitis or endotheliitis). Immunosuppression was similar in both groups. Other etiologies of graft dysfunction were excluded by ultrasound, cholangiography, and cultures. High-quality total RNA was extracted from snap frozen liver biopsies, reverse transcribed, labeled with biotin, and fragmented according to established protocol. Twenty-five genes were relatively overexpressed, and 15 were relatively underexpressed by > or = twofold in the ACR when compared with the HCV group. ACR was most notably associated with the relative overexpression of genes associated with major histocompatibility complex I and II, insulin-like growth factor-1 expression, apoptosis induction, and T-cell activation. In HCV-infected liver transplant recipients, ACR is associated with an intragraft gene expression profile that is distinct from that seen during recurrence of HCV. These experiments provide evidence that alloimmunity, as indicated by expression of T-cell activation and apoptosis-inducing genes, is less important in recurrence of HCV than in ACR. Further studies are required to determine whether gene expression profiles, either intragraft or in serum, can be used for the diagnosis and differentiation of ACR from recurrence of HCV.