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Keywords:

  • caveolin-1;
  • caveolae;
  • Type II cells;
  • electron microscopy;
  • stereology

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Caveolin-1 (Cav-1) is highly expressed in alveolar epithelial type I (AE1) and endothelial cells of the alveolar region of the lung. Interestingly, alveolar epithelial type II (AE2) cells that are progenitors of the AE1 cells do not express Cav-1. We investigated whether genetic Cav-1 deficiency alters the phenotype of AE2 cells and their microenvironment using stereology. Total number, mean volume, and subcellular composition of the AE2 cells were not altered in Cav-1−/− when compared with wild-type mice. The alveolar septa were thickened and contained a significantly greater volume of extracellular matrix. Thus, AE2 cells as progenitors of AE1 cells are not critically involved in the severe pulmonary phenotype in Cav-1-deficient mice. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

Caveolae are flask-, omega-, or cup-like plasma membrane invaginations with the shape depending on the mode of fixation (Palade and Bruns, 1968; Schlörmann et al., 2010). The existence of caveolae depends on the presence of certain structural protein components, the so-called caveolins (Cav) 1, 2, and 3. Cav-1 and Cav-2 are expressed in the same cell types, whereas Cav-3 is predominantly present in muscle cells (Tang et al., 1996; Scherer et al., 1997). A variety of functions has been attributed to caveolae, including transcytosis, signal transduction, cholesterol transport, and tumor suppression; however, the development of mice deficient in caveolins has greatly enhanced the understanding of the biological role of caveolae (reviewed in Hnasko and Lisanti, 2003).

In the alveolar region of the lung, a variety of cell types possesses a great number of caveolae including alveolar epithelial type I (AE1) cells and endothelial cells, a fact that may explain the severe pulmonary phenotype of the Cav-1 knockout (Cav-1−/−) mouse (Drab et al., 2001; Razani et al., 2001). The lungs of Cav-1−/− mice exhibit thickening of alveolar septa, increased deposition of extracellular matrix, and proliferation of endothelial cells. The nearly complete absence of caveolae in the cells of the lungs is accompanied by a strong decrease in Cav-2 protein despite normal expression at the mRNA level. In addition, Cav-1−/− mice are less tolerant to stress exercise than their wild-type (WT) littermates (Drab et al., 2001; Razani et al., 2001).

Despite the strong expression of Cav-1 in AE1 cells, their progenitor cells, the alveolar epithelial type II (AE2) cells do hardly express any Cav-1 in adult mammals (Campbell et al., 1999; Newman et al., 1999). Interestingly, Cav-1 is present in fetal AE2 cells and seems to possess a functional role in the mechanotransduction of AE2 cells (Wang et al., 2010). Besides their role as progenitors of AE1 cells, AE2 cells synthesize, store, secrete, and partly recycle pulmonary surfactant, which lowers the surface tension at the air–liquid interface and has immunomodulatory functions (reviewed in Ochs, 2010).

The transient expression of Cav-1 in AE2 cell development (Wang et al., 2010) and the hypercellular phenotype of alveolar septa in the Cav-1−/− mouse (Razani et al., 2001) led us to hypothesize that AE2 cells may also show structural abnormalities in Cav-1−/− mice. To test this hypothesis, we compared the phenotype of the alveolar septa in WT and Cav-1−/− mice with special emphasis on AE2 cells using design-based stereological methods.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Female Cav-1−/− and WT mice with a genetic background of C57BL/6 × SV129 (n = 5 each) were held under specific pathogen-free conditions and were sacrificed at the age of 15 weeks by exsanguination under isoflurane anesthesia. The lungs were fixed by tracheal instillation of a fixative containing 1.5% of glutaraldehyde and 2% of paraformaldehyde in phosphate buffer. After volume estimation by fluid displacement, systematic uniform random sampling was used to obtain samples for light microscopy (LM) and for transmission electron microscopy (TEM) according to standard procedures (Ochs, 2006). Both light and electron microscopic samples were postfixed with 1% osmium tetroxide and stained en bloc with half-saturated uranyl acetate. Dehydration via ascending ethanol (TEM) or acetone (LM) series was followed by embedding in epoxy resin or glycol methacrylate embedding (Technovit 7100, Heraeus Kulzer, Wehrheim, Germany; Mühlfeld et al., 2007). All stereological analyses at LM and TEM level conform to the guidelines set forth by the American Thoracic Society and the European Respiratory Society (Hsia et al., 2010). At the light microscopic level, point counting (Weibel, 1979) was used to estimate the volume fraction of the gas-exchange region, air-filled spaces, and alveolar septa. The surface density of the alveolar epithelium and the capillary endothelium were estimated using intersection counting (Weibel, 1979). The number-weighted mean volume and the numerical density of AE2 cells were estimated using the rotator and the physical disector (Sterio, 1984; Vedel-Jensen and Gundersen, 1993). At the TEM level, the volume densities of subcellular AE2 cell compartments (mitochondria, lamellar bodies, nucleus, and residual) and the volume densities of septal compartments (capillary lumen, endothelium, AE1 cells, AE2 cells, interstitial cells, extracellular matrix, and lipid droplets) were estimated using point counting (Weibel, 1979). All densities were multiplied by the respective reference volume to obtain total values. In addition, the volume-to-surface ratio of lamellar bodies and AE2 cell mitochondria was estimated using point and intersection counting (Weibel, 1979).

Statistical analysis was carried out applying a nonparametric two-sided Mann Whitney U test (SPSS 18.0). Differences between the two groups were regarded as significant if P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Stereological data are summarized in Table 1. Figures 1 and 2 provide representative light and electron micrographs of WT and Cav-1−/− alveolar region and AE2 cells, respectively. The volume of the lungs of the Cav-1−/− mice tended to be higher than that of controls without reaching statistical significance. Despite the tendency to higher lung volumes, the total surface area of the alveolar epithelium was similar in both groups, a fact that was achieved by a significantly increased surface density in the WT mice. Indeed, the alveolar region of the WT mice had a more compact appearance, particularly alveolar ducts appeared to be enlarged in Cav-1−/− mice. However, the overall alveolar architecture was similar between WT and Cav-1−/− mice (Fig. 1). Nevertheless, the knockout mice showed the typical thickening of alveolar septa. The latter was also reflected by the volume-to-surface ratio of the alveolar septa, which was significantly enhanced in the Cav-1−/− mice. No differences were observed in the total surface area of the capillary endothelium; however, a tendency toward a reduced surface density of the endothelium was found in the Cav-1−/− mice.

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Figure 1. Light micrographs displaying the architecture of the alveolar region of the lung in wild-type (A) and Cav-1−/− mice (B).

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Figure 2. Electron micrographs displaying the morphology of AE2 cells in the lungs of wild-type (A) and Cav-1−/− mice (B).

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Table 1. Summary of stereological data and statistical analysis
 WTCav-1−/−P value
  1. Abbreviations: V, volume; S, surface area; N, number; VV, volume density; νN, number-weighted mean volume; VS ratio, volume to surface ratio; air, air-filled spaces (alveolar lumina); par, parenchyma; sept, alveolar septal tissue; AEpi, alveolar epithelium; AEnd, alveolar capillary endothelium; AE2, alveolar epithelial type II cell; AE1, alveolar epithelial type I cell; LB, lamellar body; mito, mitochondria; Caplum, capillary lumen; IC, interstitial cell; ECM, extracellular matrix; LD, lipid droplets.

V(lung) (mm3)708 ± 45891 ± 1420.151
V(air,par) (mm3)393 ± 66480 ± 1000.31
V(sept,par) (mm3)186 ± 20240 ± 300.008
S(AEpi,lung) (cm2)547 ± 61525 ± 850.69
S(AEnd,lung) (cm2)238 ± 43216 ± 430.841
V(sept)/S(AEpi) (μm)3.43 ± 0.494.60 ± 0.340.008
N(AE2,lung) (×106)5.29 ± 1.195.39 ± 0.950.548
νN (AE2) (μm3)545 ± 42532 ± 470.841
VV(LB/AE2) (%)19.67 ± 2.7116.37 ± 3.130.095
V(LB,AE2) (μm3)107.9 ± 20.987.2 ± 19.70.095
V(LB,lung) (mm3)0.571 ± 0.1840.458 ± 0.0700.31
VS ratio(LB) (μm)0.248 ± 0.0300.215 ± 0.0200.151
VS ratio(mito) (μm)0.161 ± 0.0180.162 ± 0.0141
V(LB)/S(AEpi) (mm3/mm2)10.5 ± 3.38.9 ± 1.70.69
V(Caplum,sept) (mm3)63.4 ± 6.369.7 ± 7.00.222
V(AEnd,sept) (mm3)35.4 ± 7.642.3 ± 8.00.222
V(AE1,sept) (mm3)32.6 ± 1.940.7 ± 6.80.095
V(AE2,sept) (mm3)15.5 ± 5.820.8 ± 4.00.095
V(IC,sept) (mm3)25.0 ± 3.133.8 ± 8.40.151
V(ECM,sept) (mm3)13.6 ± 1.432.6 ± 9.20.008
V(LD,sept) (mm3)2.11 ± 0.662.72 ± 1.051

At the electron microscopic level, the alveolar septa of Cav-1−/− mice appeared enlarged with focal areas of large deposits of extracellular matrix, particularly collagen fibrils were abundantly observed. However, in other areas, the alveolar septa appeared widely normal. The thickening of the alveolar septa was reflected by a tendency of epithelial, endothelial, and interstitial cell volumes to higher values. The increase in total cellular volumes did not reach statistical significance with the means of all cell types being 20%–40% higher than in the control group. In contrast, the volume of the extracellular matrix was 2.35 times higher in the Cav-1−/− than in the WT mice. In both groups, lipid droplets were frequently observed in interstitial cells of alveolar septa. No quantitative difference regarding the total volume of this organelle was observed between the groups. The further analysis of the AE2 cells revealed that their mean volume and their total number did not differ between WT and Cav-1−/− mice. Although there was a trend toward a lower volume of smaller lamellar bodies, this was not supported by the qualitative analysis or the total values (Fig. 2). Thus, the intracellular surfactant reserve per unit of alveolar surface area is not changed in the AE2 cells of Cav-1−/− mice. Additional parameters to characterize AE2 cells like mitochondrial volume or volume-to-surface ratio did not reveal any differences in the phenotype of these cells in Cav-1−/− mice.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Caveolae are abundantly found in the alveolar region of the lungs, particularly in the alveolar epithelium and capillary endothelium. Among the two alveolar epithelial cell types, the Type I cells strongly express caveolin-1 (Cav-1), the major structural protein component of caveolae, and contain a great number of caveolae; however, the Type II cells only transiently express Cav-1 during development. Therefore, caveolae are absent from adult AE2 cells. Disruption of the Cav-1 gene causes the complete absence of caveolae in the cells of the alveolar region of the lungs and a severe pulmonary phenotype with hypercellular, thickened alveolar septa (Drab et al., 2001; Razani et al., 2001).

However, the biology of AE1 and AE2 cells is closely linked because of the role of AE2 cells as progenitors of AE1 cells. This as well as the AE2 cells being producers of surfactant has led to the view that these cells defend the alveolar region in many different ways (Fehrenbach, 2001). Therefore, AE2 cells and the surfactant system are crucial antagonists against the pathogenic mechanisms of acute lung injury. As Cav-1 has been shown to have a variety of functions in processes such as endothelial cell apoptosis, chemotaxis, and polymorphonuclear cell adhesion (Jin et al., 2011), it is important to understand whether Cav-1 is necessary for AE2 cell homeostasis. Therefore, in the current study, we aimed at analyzing the phenotype of AE2 cells in relation to their altered microenvironment in the Cav-1−/− mouse lung. This study is the first that provides quantitative morphological data on the phenotype of the Cav-1−/− mouse lung.

The stereological data of the current study confirm the originally described phenotype of the Cav-1−/− mouse lung (Drab et al., 2001; Razani et al., 2001) in that the thickening of the alveolar septa is reflected by an increased volume of septal tissue, an increased volume-to-surface ratio of the septa, and by increases in the volumes of the septal components. Qualitatively, a mild enlargement of alveolar ducts was observed in the knockout mice that may add to the trend to higher lung volumes in these mice. Such changes are described to be present in chronic lung inflammation and emphysema (Xisto et al., 2005; Tuder et al., 2006); however, there was no sign of chronic inflammation in Cav-1−/− mice. The increased septal tissue volume, the changes in alveolar duct size, and the tendency toward higher lung volume in the Cav-1−/− mouse, however, are not associated with an increase in the surface area of the functionally relevant alveolar epithelium or capillary endothelium. Thus, the total gas exchange surface area is not altered in Cav-1−/− mice. As the oxygen diffusion capacity scales with the surface area and scales inversely with the diffusion distance (Weibel, 1972), the deposition of extracellular matrix and the thickening of septa increases the oxygen diffusion distance, which—beyond changes in pulmonary mechanics (Le Saux et al., 2008)—adds to the decreased tolerance to exercise stress observed for this genotype.

Alveolar septa contain two cell types that have a highly active lipid metabolism, namely lipofibroblasts and AE2 cells. Although most of the lipids in AE2 cells are phospholipids related to surfactant metabolism (Ochs, 2010), lipofibroblasts contain lipid droplets involved in retinoic acid storage and are necessary for the regulation of alveolar septa during development (Torday and Rohan, 2007). Nevertheless, the lipid metabolism of both cell types is closely linked (Torday and Rohan, 2011) and significant changes in one of the cell types should affect the other cell type as well.

As Cav-1−/− mice have defects in lipid droplet formation (Cohen et al., 2004), we investigated the volume of lipid droplets in alveolar septa. In WT and Cav-1−/− mice, lipid droplets were nearly exclusively present in lipofibroblasts and only rarely observed in AE2 cells. The total volume of lipid droplets was not different between both groups. In AE2 cells, the main surfactant storing organelles are the lamellar bodies, which appeared to be slightly smaller in the AE2 cells of Cav-1−/− mice. However, when the total volume of lamellar bodies in the lung was calculated, no difference became apparent between WT and Cav-1−/− mice. The ratio between lamellar body volume and alveolar epithelial surface area also remained constant and within a normal range (Wirkes et al., 2010). Thus, the reserve of surfactant does not seem to be altered in the Cav-1−/− mouse. Although the intra-alveolar surfactant pool is more directly important for pulmonary mechanics, our data suggest that defects in pulmonary surfactant are not likely to be involved in the mechanical dysfunction observed in Cav-1−/− mice (Le Saux et al., 2008). In addition, the ultrastructure of AE2 cells did not show any abnormalities in the Cav-1−/− mouse, and all stereological parameters estimated for the knockout mouse were in the same range as the WT data. Therefore, we conclude that Cav-1 deficiency does not have major effects on AE2 cell structural properties and surfactant homeostasis. As Cav-1 deficiency rather enhances the number of AE1 cells, a higher number of AE2 cells as their progenitors does not seem to be necessary. However, given the abnormal morphology of alveolar septa and the hyperpermeability associated with Cav-1 deficiency, the role of the surfactant system might require further functional studies to understand whether Cav-1 plays a role in surfactant metabolism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Tamara Papadakis, Gerd Magdowski, and Gerhard Kripp for expert technical assistance with the preparation of the microscopic sections.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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