The broad goal of the alveolar tissuegenesis project is to establish the model of bleomycin induced acute lung injury in C57/BL6 mice and to demonstrate that airway delivery of freshly isolated type II alveolar epithelial cells from normal mice will reduce the lung injury in the bleomycin treated mice. The proposal is as follows. Acute lung injury is a major cause of morbidity and mortality in both children and adults. The pathologic hallmark of acute lung injury is diffuse alveolar damage. The damage is a direct result of injury to the alveolar epithelial cells and capillary endothelial cells maintaining the alveolar capillary barrier. The alveolar epithelium appears to be especially important as a site of injury as it is primary responsible for maintaining the integrity of the alveolar capillary membrane as well as synthesizing and processing of alveolar surfactant phospholipids and surfactant associated proteins. Various agents have been used to model acute lung injury including such as bleomycin that is used in cancer chemotherapy, hyperoxia which is commonly required to maintain normoxemia in critically ill patients, and pulmonary specific toxins such as the herbicide paraquat. Some agents such as hyperoxia predominantly damage the pulmonary capillary endothelial cell;whereas, other agents such as paraquat may predominantly damage the type I and type II alveolar epithelial cells. In practice, most agents that result in acute lung injury damage both the alveolar epithelium and the pulmonary capillary endothelium. Whereas injury occurs to both capillary endothelial cells and type I and type II alveolar epithelial cells during acute lung injury, it is the loss of the alveolar epithelial cells that appears to be critical for destruction of the alveolar capillary units and subsequent development of fibrosis (scarring of lung tissue). One approach to potentially reverse the catastrophic events associated with acute lung injury is to restore the alveolar capillary unit and its barrier function. Molecular and celular mechanisms associated with pulmonary fibrosis are still obscure. Apoptosis has been thought to be a non-inflammatory means of removing injurious cells thus facilitating lung injury. Mitochondria play an important role in mediating apoptosis thus functional understanding of mitochondria during oxidative stress secondary to bleomycin will help us to design a better model for lung injury and repair. The injurious effects of inflammatory cells such as neutrophils has been implicated in lung injury and yet remains poorly understood. Restoration of alveolar type II epithelial cells could help restore the normal synthesis and processing of pulmonary surfactant and surfactant apoproteins, which would be especially important for premature infants with insufficient pulmonary surfactant production. The type II cell is in the lung and also is considered the progenitor of the type I cell which is important for gas exchange. It is possible early restoration of type II cells might preclude further lung inflammation and reduce the risk of state of chronic injury and repair. Recent studies indicate that during acute lung injury from bleomycin, marrow-derived progenitor cells administered intravenously can migrate to the lung and change into alveolar epithelial cells. It remains unclear whether this process of engraftment can actually reduce acute lung injury and even mortality. This is a very important question to answer. Since our laboratory has significant experience with alveolar epithelial cells, acute lung cell injury and bleomycin toxicity animal models, we are well suited to investigate the therapeutic potential of tissuegenesis initiated by the delivery of type II cells. While the mechanisms of bleomycin-induced lung cell toxicity have been well studied, current therapy to reduce the lung toxicity is inadequate and novel therapeutic approaches for affected patients need to be developed. Our laboratory has recently demonstrated the feasibility of airway delivery of genetically engineered macrophages to the alveolar structures to modify the alveolar environment (PNAS 98:14589-94, 2001). We propose that airway delivery of type II cells or other potential progenitor cells during bleomycin lung injury will result in successful engraftment of these cells onto the injured alveolar surface. This is the first study to our knowledge where airway delivery of cells has been used therapeutically to alter the alveolar environment. If successful, these studies would represent a new approach to alveolar tissue repair during acute lung injury and would be potentially applicable to treat acute lung injury in children and adults. Hypothesis: Airway delivery of alveolar epithelial progenitor cells to the alveolar structures during bleomycin lung injury will result in engraftment and replacement of the injured alveolar epithelium and will hasten recovery of lung function and increase survival. Primary Specific Aims: 1.To determine mechanisms of bleomycin mediated injury to alveolar epithelial cells. 2.To demonstrate that delivery of alveolar epithelial progenitor cells (e.g. type II alveolar epithelial cells) in a mouse model will localize to the alveolar structures and engraft to the injured alveolar surface during bleomycin-induced acute lung injury. 3.To determine if successful engraftment of alveolar epithelial progenitor cells to the alveolar epithelium will hasten recovery from bleomycin-induced acute lung injury and increase survival. In the past year since we arrived at NIEHS, we have established that our lab can recreate the injury model by bleomycin and that we can successfully isolate the type II cells in sufficient purity and viability to conduct the proposed studies. Additionally we have enhanced our ability to quantify bleomycin-induced lung injury using computer-assisted morphometric analysis with Metamorph which will permit for the first time a quantitative approach to bleomycin injury at baseline as well as to assess the impact of donor type II cells as repair cells. Furthermore, we have also demonstrated how bleomycin damages alveolar epithelial cells by oxidative damage to mitochondrial DNA which is another first in bleomycin injury. Finally we have developed tools in the laboratory that allow us to precisely determine the emigration of inflammatory cells such as neutrophils in to the lung following bleomycin injury. These studies will enhance our approach to reducing injury and improving repair following exposure to bleomycin.
| Pasula, Rajamouli; Britigan, Bradley E; Turner, Joanne et al. (2009) Airway delivery of silica increases susceptibility to mycobacterial infection in mice: potential role of repopulating macrophages. J Immunol 182:7102-9 |
