Inhibition of neuroretinal cell death by insulin-like growth factor-1 and its analogs.

PURPOSE: Visual loss secondary to retinal ischemia/hypoxia can be a serious complication of diabetic retinopathy, as well as other vascular insults. We used R28 retinal precursor cells, as well as primary rat retinal cell cultures, to test whether the neuroprotective growth factor IGF-1 would protect retinal cells from dying under conditions of hypoxia or serum-starvation. We also utilized three IGF-1 analogs ([LongR3], [Ala31], and [Leu24][Ala31]) with altered affinities for the IGF-1 receptor and/or IGF-1 binding proteins in order to address the mechanism(s) of IGF-1 neuroprotection. METHODS: Retinal cultures were subjected to hypoxia (95% N2/5% CO2 for 0-8 h), or serum-starvation (0% serum for 48 h). Experimental cultures were pre-treated for 24 h with 0-100 ng/ml of IGF-1 or its analogs. Retinal cultures were analyzed for the extent of cell death by trypan blue exclusion assay, TUNEL in situ, as well as ssDNA analysis specific for apoptosis. RESULTS: IGF-1 and all three IGF-1 analogs tested were able to inhibit neuroretinal cell death at a concentration of 50 ng/ml. Neuroprotection was evident under conditions of hypoxia or serum-starvation. CONCLUSIONS: IGF-1, as well as IGF-1 analogs, improves survival of neuroretinal cells in vitro, under conditions of hypoxia or serum-starvation. Since all three IGF-1 analogs inhibit cell death to some degree, we interpret these results to mean that IGF-1-mediated inhibition of cell death does not depend upon strong affinities for the IGF-1 receptor or IGF-1 binding proteins. Further studies will reveal additional information as to the pathways responsible for IGF-1-mediated neuroprotection of retinal cells.

Discordant pulmonary proinflammatory cytokine expression during acute hyperoxia in the newborn rabbit.

Newborn animals are resistant to oxygen toxicity. To investigate this phenomenon, the proinflammatory cytokines interleukin (IL)-1 beta, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were measured during newborn rabbit hyperoxic lung injury. Pups were exposed to > 95% O2 for 8-9 days, followed by 60% O2 until 36 days of age. Lung lavage fluid, RNA, and tissue sections were collected at 0, 2, 4, 6, 8, 10, 12, 14, 22, and 36 days. Acute inflammation occurred by 6-10 days of hyperoxia, and fibrosis by 22 days. Northern hybridization of lung homogenates from hyperoxia-exposed pups showed elevated MCP-1 and IL-8 mRNA expression at 6 and 10 days, respectively, compared to age-matched, air-exposed controls. Lavage fluid IL-8 protein also peaked at 10 days, and was strongly correlated to neutrophil numbers in lavage. In situ hybridization revealed elevated IL-1 beta mRNA in macrophages, alveolar epithelial and interstitial cells at 2-10 days, elevated MCP-1 mRNA in similar cell types at 4-8 days, and elevated IL-8 mRNA in these cells and neutrophils at 4-10 days. IL-1 beta and IL-8 expression peaked during peak inflammation, whereas peak MCP-1 expression preceded macrophage influx. Comparing newborn and adult animals' chemokine response may help explain their differences in hyperoxia susceptibility.

Hyperoxia increases keratinocyte growth factor mRNA expression in neonatal rabbit lung.

Acute hyperoxic lung injury remains a major factor in the development of chronic lung disease in neonates. A critical step in the repair of acute lung injury is the proliferation of type II alveolar epithelial cells. Type II cell proliferation is stimulated by keratinocyte growth factor (KGF), an epithelial cell-specific mitogen. We sought to investigate KGF mRNA expression in relation to type II cell proliferation during hyperoxic lung injury. We studied a previously described newborn (NB) rabbit model of acute and chronic hyperoxic injury [C. T. D'Angio, J. N. Finkelstein, M. B. LoMonaco, A. Paxhia, S. A. Wright, R. B. Baggs, R. H. Notter, and R. M. Ryan. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L720-L730, 1997]. NB rabbits were placed in 100% O2 for 9 days and then recovered in 60% O2. RT-PCR was used to synthesize and amplify a 267-bp fragment of rabbit KGF cDNA from whole lung RNA. KGF mRNA expression was analyzed by ribonuclease protection assay, and mRNA abundance was quantified by phosphorimaging. Proliferating cell nuclear antigen immunohistochemistry was used on lung sections to identify proliferating cells. The rabbit partial cDNA sequenced was >95% homologous to human cDNA, and all amino acids were conserved. Whole lung KGF mRNA expression was increased 12-fold after 6 days of hyperoxia compared with control lungs, and remained increased throughout the 100% O2 exposure period. Proliferating cell nuclear antigen immunohistochemistry showed an increase in type II cell proliferation after 8-12 days of hyperoxia. NB rabbits exposed to hyperoxic injury exhibit increased whole lung KGF mRNA expression preceding type II cell proliferation. KGF may be an important mitogen in the regulation of alveolar epithelial repair after hyperoxic lung injury.

Cell-specific gene expression reveals changes in epithelial cell populations after bleomycin treatment.

Epithelial repair following acute lung injury involves proliferation and differentiation of existing Clara cells and type II cells. Other mechanisms of epithelial repair may be involved in particularly severe cases. We used epithelial cell-specific markers to examine changes in the mouse lung epithelium 28 days after bleomycin treatment. The spatial distribution of surfactant proteins A, B, C (SPA, SPB, SPC), and Clara cell-specific protein (CC10) mRNA was compared by in situ hybridization in serial lung sections. CC10 mRNA-containing airway cells were replaced in many areas by SPB mRNA-expressing, ciliated cells that did not contain CC10 mRNA. In distal airway regions, we observed a subpopulation of epithelial cells that appeared to express SPA, SPB, SPC, and CC10 mRNA, and speculated that they may represent a multipotential stem cell population. These cells were found in focal clusters, which suggests that they expanded from a common cell. CC10 mRNA-containing cells were seen in alveolar-like structures thought to be the result of Clara cell migration or outpocketing. Our data suggest that there are repair mechanisms involved in epithelial repair after severe injury that have not previously been described.

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

Electron microscopic identification and morphologic preservation of enriched populations of lung cells isolated by laser flow cytometry and cell sorting: a new technique.

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).

Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC.

We have determined that murine lung fibroblasts are divisible into two major subpopulations based on expression of Thy 1. Twenty-four to fifty-three percent of freshly isolated lung cells displayed Thy 1 and were separated using FACS into Thy 1+ and Thy 1- fractions for morphologic examination. Analysis by electron microscopy revealed that both the Thy 1+ and Thy 1- fractions contained fibroblasts. Freshly isolated lung cells cultured for 2 wk consisted of greater than 95% fibroblasts, with 28 to 49% displaying Thy 1. These cells were sorted by FACS into Thy 1+ and Thy 1- lines that maintained a stable phenotype over many weeks and that were used as a source to obtain stable fibroblast clones. Adherent pulmonary fibroblasts are not phagocytic and lack the markers of macrophages, dendritic cells, B lymphocytes, and T lymphocytes (with the exception of Thy 1). Interestingly, the Thy 1- fibroblasts spread more and contained a more extensive microfilament and microtubule network than did the spindly and often lipid-containing Thy 1+ population. Both populations of fibroblasts synthesized collagen. Class I MHC expression was very low on Thy 1+ and Thy 1- fibroblasts, but high levels were displayed after gamma-IFN treatment. Most exciting was the unexpected finding that only the Thy 1- lines and clones displayed class II MHC (Ia) in response to treatment with gamma-IFN. Moreover, only the Thy 1- fraction (gamma-IFN-treated) presented antigen to T lymphocyte clones, an observation that suggests that this subset of cells may be involved primarily in promoting chronic lung inflammation, which is associated with developing fibrosis. Thus, two populations of pulmonary fibroblasts exist, defined by the expression of Thy 1, distinguishing morphology, inducibility for Ia expression, and antigen-presenting function. It should now be possible, using these characteristics, to ascertain the role of pulmonary fibroblast subpopulations in developing fibrosis.