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Keywords:

  • alveolar growth;
  • lung development;
  • ozone;
  • allergen;
  • rhesus monkey

Abstract

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

Exposure to oxidant air pollutants in early childhood, with ozone as the key oxidant, has been linked to significant decrements in pulmonary function in young adults and exacerbation of airway remodeling in asthma. Development of lung parenchyma in rhesus monkeys is rapid during the first 2 years of life (comparable to the first 6 years in humans). Our hypothesis is that ozone inhalation during infancy alters alveolar morphogenesis. We exposed infant rhesus monkeys biweekly to 5, 8hr/day, cycles of 0.5 ppm ozone with or without house dust mite allergen from 1 to 3 or 1 to 6 months of age. Monkeys were necropsied at 3 and 6 months of age. A morphometric approach was used to quantify changes in alveolar volume and number, the distribution of alveolar size, and capillary surface density per alveolar septa. Quantitative real time PCR was used to measure the relative difference in gene expression over time. Monkeys exposed to ozone alone or ozone combined with allergen had statistically larger alveoli that were less in number at 3 months of age. Alveolar capillary surface density was also decreased in the ozone exposed groups at 3 months of age. At 6 months of age, the alveolar number was similar between treatment groups and was associated with a significant rise in alveolar number from 3 to 6 months of age in the ozone exposed groups. This increase in alveolar number was not associated with any significant increase in microvascular growth as measured by morphometry or changes in angiogenic gene expression. Inhalation of ozone during infancy alters the appearance and timing of alveolar growth and maturation. Understanding the mechanism involved with this altered alveolar growth may provide insight into the parenchymal injury and repair process that is involved with chronic lung diseases such as severe asthma and COPD. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

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

Humans are exposed to a variety of inhaled infectious diseases and irritants such as air pollutants and environmental tobacco smoke that damage the lung, thus it is critical that we understand the nature of these insults on the developmental process of the lung. The synergistic effect of ozone and allergen exposure during early development to promote airway wall remodeling has been well established, however, the effect on alveolar growth and development is not known (Miller et al., 2003). In previous work, we have demonstrated that episodic ozone exposure amplifies the allergic airway response in a monkey model of asthma (Schelegle et al., 2003a, b). Using a similar monkey model, we set out to establish the effect of episodic ozone (0.5 ppm) and allergen exposure on alveolar growth and development. Alveolarization, by formation of secondary interalveolar septa (IAS), occurs from 36 weeks of gestation to 1–2 years of age while microvascular maturation, by remodeling of IAS and restructuring of the capillary bed, occurs from birth to 2–3 years of age (Schelegle et al., 2003a, b; Miller et al. 2003; Ochs et al., 2004). This process of alveolarization continues in the postnatal period to reach an average of 450 million alveoli in the human adult (Ochs et al., 2004). When lung morphology of rhesus macaques and humans is compared, there are similarities in the segmental arrangement, the structure and branching pattern of airways, and arterial structure after birth (Tyler et al., 1991; McBride, 1991). The general developmental stages in the rhesus monkey are as follows: embryo, 21–45 days gestation; fetus, 45–165 days gestation; newborn, 24 h postnatal; neonate, 0–1 month; infant, 1–12 months; juvenile, 12–24 months; adolescent, 2–4 years; and young adult, 4–8 years (Golub et al., 1984; Leek et al., 1984). In previous work, we observed a rapid alveolar growth phase during the first year and a slower growth phase that continued until full somatic growth was accomplished at approximately 7 years of age (Hyde et al., 2007). Using a similar experimental design, we exposed rhesus monkeys during the first year of life to cyclic ozone and house dust mite allergen (HDMA) to examine potential alterations in parenchymal growth. The principle aim of this study was to measure the stress response of episodic ozone and allergen exposure on postnatal alveolarization, alveolar capillary surface density, and gene expression of candidate gene associated with angiogenesis in the lung.

METHODS

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

All monkeys selected for these studies were California National Primate Research Center (CNPRC) colony-born rhesus macaques (Macaca mulatta). Care and housing of animals complied with the provisions of the Institute of Laboratory Animal Resources and conformed to practices established by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animal studies conformed to applicable provisions of the Animal Welfare Act and other federal statutes and regulations relating to animals (Guide for the Care and Use of Laboratory Animals; National Institutes of Health, revised 1985). Experimental protocols were reviewed and approved by the University of California, Davis Institutional Animal Care and Use Committee. Male rhesus monkeys (Macaca mulatta) were housed in 4.2 m3 capacity exposure chambers within the CNPRC. Infants were removed from the mothers at birth and placed in a nursery for bottle feeding and 24 hr care. At 2 days of age, animals were selected randomly, assigned to groups, and placed in a nursery setting. Chambers were ventilated at a rate of 30 changes per hour with filtered air (FA). Each exposure chamber housed three animals throughout the study. Animals were selected for the study based on physical examination and negative intradermal skin test reactivity to HDM. Body weight between groups at 2 days of age was comparable.

Animals on the study were monitored by trained CNPRC animal care, research, and veterinary staff. Highly trained CNPRC nursery technicians provide animal care and health monitoring as follows: Official health observations are recorded twice daily. Infant temperatures, weights, and food intakes are recorded according to established CNPRC nursery-rearing standard operating procedures. In addition, nursery personnel carefully observe the subjects throughout the day and evening as they provide animal care. Any abnormalities are immediately reported to the CNPRC veterinary staff. During HDMA exposures, research personnel carefully monitor animals, with respiratory rates recorded at key time points before and during exposures. Any abnormalities are reported immediately to the CNPRC veterinary staff. CNPRC veterinary staff regularly assesses study animals and respond to any daily health concerns or reports. CNPRC veterinary staff provided treatment or intervention as clinically indicated in consultation with the principal investigator.

Rhesus monkeys were sensitized to HDMA [Dermatophagoides farina (HDMA)], ozone (O3), and HDMA + O3 groups at 14 days of age by subcutaneous inoculation (SQ) of HDMA in alum and intramuscular injection of heat-killed Bordetella pertussis cells and again at 28 days of age by SQ of HDMA in alum. The HDMA was purchased from Greer Laboratories. (Lenoir NC) and was used for both SQ inoculation and aerosol administration. HDMA sensitization was confirmed via skin testing with intradermal HDMA on Day 38 of the exposure protocol. At 1 month of age 48 infant rhesus monkeys (30 days old) were exposed to 5 or 11 episodes of FA (n = 12), HDMA aerosol (n = 12), ozone (O3)(n = 12), or HDMA + O3(n = 12) (5 days followed by 9 days of FA for a total of 2 weeks). HDMA was delivered as an aerosol on days 3–5 for 2/hr (n = 24). Ozone was delivered for 8 hr/day at 0.5 ppm for 5 contiguous days (n = 24). Twenty-four of the monkeys (HDMA and HDMA + O3 groups) were sensitized to HDMA (Dermatophagoides farinae) at 14 days of age by SQ of HDMA in alum and intramuscular injection of heat-killed Bordetella pertussis cells and again at 28 days of age by SQ of HDMA in alum. HDMA sensitization was confirmed via skin testing with intradermal HDMA on Day 38 of the exposure protocol (Fig. 1). Details of ozone and HDMA exposure methods for this study were previously reported. In brief, ozone was generated as previously described and concentration was monitored using a Dasibi 1003-AH ozone analyzer (Dasibi Environmental Corporation, Glendale, CA) (Wilson et al., 1984; Schelegle et al., 2003a, b). Although ozone levels as low as 0.15 ppm induce pathologic changes in the central acinus in rhesus monkeys and the National Air Quality Standard is currently at 0.075 ppm, we chose an ozone level of 0.5 ppm to enhance the ozone effect on the developing rhesus lung (Harkema et al., 1993). HDMA aerosol exposures were generated using HDM extract (Greer Laboratories, Lenoir, NC) diluted in phosphate-buffered saline (PBS) as described previously (Schelegle et al., 2001). During exposure, air samples were drawn from the chamber and the mass concentration, protein content, and aerodynamic size distribution characterized as previously described (Schelegle et al., 2001). This exposure protocol is summarized in Tables 1 and 2.

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Figure 1. Exposure diagram. Timing of HDMA and Ozone exposure in a typical 14-day cycle.

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Table 1. Ozone concentrations in chambers during exposure of infant rhesus monkeys
 ppm Ozone 
GroupMean±SDMinimumMaximumNumber of Samples
1999
 Ozone0.5000.0050.4440.5286394
 Allergen and Ozone0.5000.0050.4640.5296532
2000
 Ozone0.4990.0050.4500.5206394
 Allergen and Ozone0.4990.0050.4710.5236532
Table 2. House dust mite allergen aerosol in chamber during exposure of infant rhesus monkeys (1999 and 2000)
Aerosol concentrations from filter samplesNumber of samples
  • a

    Log-normal distribution fitted to chloride content of stages and after-filter.

  • b

    Mass median aerodynamic diameter.

  • c

    Geometric standard deviation.

Amino acid analysis (μg/m3 ± SD protein)435 ± 9620
Total mass (mg/m3 ± SD)7.39 ± 0.59150
Aerodynamic size from cascade impactor samplesa 
  MMADb (μm ± SD)1.29 ± 0.1211
  σgc ± SD2.12 ± 0.2611

Monkeys were necropsied at 14 (5 cycles) (n = 6/group) or 26 weeks (11 cycles) (n = 6/group). In our experience, the right middle lobe is representative of the whole lung. Using alveolar number, variance among lobes of the left lung was of the same order as the variance among right lungs in rhesus monkeys. (Hyde et al., 2004).The right middle lung lobes were fixed via the airways through a tracheal cannula with 1% glutaraldehyde-1% paraformaldehyde in cacodylate buffer (330 mosM, pH 7.4) at 30-cm fluid pressure (8 hr). After fixation, lobes were embedded in 4% agar, isotropically oriented using an orientator, sliced into 5-mm slabs, and sampled using a smooth fractionator (Mattfeldt et al., 1990; Gundersen, 2002). The sampled tissue was embedded in paraffin, cut in 5-μm serial sections, and stained with hematoxylin and eosin. Lobe volume (VL) without the trachea and extrapulmonary bronchi was estimated by its buoyant weight in PBS (Golub et al., 1984a, b). Stratified random sampling was use to collect fields from sections along with a double lattice test system of 25/100 points to evaluate the volume densities (Vv) of parenchymal and nonparenchymal components using point counting and the absolute volumes of parenchyma (Vpar), alveoli (Valv), interalveolar septa (Vias), alveolar duct core air (Vad), and nonparenchyma (Vnp) were determined by multiplying their Vv by VL in units of cm3. Alveolar numbers were estimated by counting the number of entrance rings in paired H&E sections by the disector technique of counting bridges between sections demonstrated in Fig. 2 (Hyde et al., 2004; Ochs et al., 2004). Alveolar volumes (number-weighted) were calculated directly from cumulative alveolar volume divided by the total alveolar number in the lung, whereas a point-sampled intercept method was used to estimate volume-weighted alveolar volumes. Using both alveolar volumes, we calculated the coefficient of variation of the distribution of number-weighted alveolar volumes (Gundersen and Jensen, 1985). To identify surface density of pulmonary capillaries, a fluorescent approach utilizing goat anti-human CD31 (BD Pharmingen) combined with secondary antibody, anti-goat Alexa 488 (Life Technologies, Grand Island, NY) was used. The surface density of septal (pulmonary) capillaries and IAS in the right middle lung lobe were estimated using point and intersection counting and expressed as a ratio of capillary to IAS surface (Hsia et al., 2010).

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Figure 2. Alveolar counting method. Counting of alveolar bridges (B) in serial sections where connecting bridges (B) between serial sections are demonstrated.

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Gene Expression

RNA was isolated from caudal lobes using the previously described Trizol method or the commercially available kit from Qiagen. (Alameda, Ca.). Once the RNA was isolated and quantified, cDNA was synthesized using a commercial kit from Invitrogen. (Carlsbad, Ca.) (Chomczynski and Sacchi, 2006). The relative gene expression of p311 was performed in Dr. Thiennu Vu's laboratory at UCSF using a previously published protocol (Zhao et al., 2006). The primers and probes for two distinct VEGF genes (Hs99999070_m1 and Hs0900055_m1) were purchased from ABI (Carlsbad, Ca.). The protocol for the remaining genes including the development of primers was performed in the Analytical and Resources Core of the CNPRC (http://www.cnprc.ucdavis.edu/research/arc.aspx).

Statistical Methods

We analyzed data for overall effects of exposure or age by two-way ANOVA and for differences between individual groups by one-way ANOVA combined with Fisher's least significant difference test (SYSTAT 12, SPSS, Chicago, IL). Significance was accepted at P ≤ 0.05.

RESULTS

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

Changes in Lung Volume

Representative micrographs of the gross changes in alveolar number and size are represented in Fig. 3. The effect of age and exposure on the right middle lobe volume is summarized in Table 3. Age had a much greater effect than exposure on lobe volume. As would be expected, lung volume increases with age. The distributions of lobe volumes per exposure are represented as means with standard deviations in Fig. 4. After 3 months, there is no significant difference in lobe volumes between the four different exposure groups. However, the combined exposure group of HDMA and ozone had a mean lobe volume that was statistically larger than the FA group at 6 months. To compare ozone exposure versus HDMA, we combined the groups that had HDMA exposure (HDMA + HDMA/ozone) and compared theme to ozone treated groups (ozone + HDMA/ozone). Using a two-way ANOVA approach at each age point revealed that ozone had a much stronger effect on lobe volume at the 6-month of age time point (Table 3).

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Figure 3. Representative images from each exposure cohort. (A) FA (B) HDMA (C) Ozone (D) Ozone + HDMA

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Figure 4. Lung volume. The volume of the right middle lung lobe for four treatment groups at 3 and 6 months is represented. There was a significant increase (*) (P ≤ 0.05) between the O3 + HDMA and FA groups at 6 months of age. Volume significantly increased from 3 to 6 months within the HDMA, O3, and HDMA + O3 groups (**) (P ≤ 0.05).

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Table 3. Statistical summary of exposure and age effects on alveolar growth and size
      Two-way ANOVA
 ExposureAgeHDMAOzoneHDMA + OzoneHDMA(HD) vs. Ozone
3 months6 months3 months6 months3 months6 monthsAge (months) in parenthesis HD OZONE
Lobe VolumeNSP < 0.001NSNSNSNSNS0.026NS0.009(6)
Number Alveoli0.020P < 0.001NSNS0.041NS0.006NSNSP < 0.001(3)
Number-weighted alveolar volumeP < 0.001NSNSNS0.026NS0.002NSNSP < 0.001(3), P < 0.001(6)
CvP < 0.001NSNSNS0.017NS0.001NSNSP < 0.001(3), P < 0.001(6)
Ss CapillaryP < 0.001P < 0.001NSNSP < 0.001NS0.001NSNSP < 0.001(3)

Changes in Alveolar Number and Volume

Table 3 demonstrates that age had a greater effect on alveolar number while exposure had more influence on alveolar size. The mean alveolar number difference between the exposure groups is represented in Fig. 5. After 3 months of exposure, the ozone alone and HDMA/ozone combined exposure groups had significantly fewer alveoli than the FA controls at 3 months. At the 6-month time point, the total number of alveoli is statistically greater for all the HDMA, Ozone, and HDMA/O3 groups compared to their 3-month values and there is no significant difference in number of alveoli between the exposure groups at the 6-month time point (Fig. 5). The total number of alveoli increased for the FA group from 3 to 6 months but it was not statistically different. Accordingly, the number of alveoli significantly increased from 3 to 6 months within the Ozone and HDMA/Ozone combined exposure groups. As compared with the lung volumes, analysis of ozone versus HDMA effects revealed that ozone effect on alveolar number was much greater at the 3-month time point.

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Figure 5. Alveolar number. The number of alveoli in the right middle lung lobe for four treatment groups at 3 and 6 months is represented. There was a significant decrease (*) (P ≤ 0.05) between the O3 and O3 + HDMA compared to the FA groups at 3 months of age. From 3 to 6 months alveolar number increased significantly within the HDMA, O3, and O3 + HDMA groups.

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Although the alveolar number was decreased among the exposure groups involving ozone, their respective size was much greater. Figure 6 displays the relative differences in alveolar volume between the exposure groups at 3 and 6 months. The number-weighted alveolar volume is significantly larger for the ozone groups at 3 months returning to a smaller volume by 6 months (stats). There is no statistical difference in alveolar volume between groups at 6 months, although the decrease in alveolar volume from 3 to 6 months was significant within the ozone and HDMA/ozone combined groups. The dynamic change in alveolar size between 3 and 6 months among the ozone and HDMA/ozone groups is fairly uniform and is reflected in Fig. 7, which demonstrates that the variability in alveolar size is decreased in the ozone exposure groups compared to the FA group. Compared with alveolar number and lung volume, the exposure effects are more significant for ozone exposure compared to HDMA at both the 3- and 6-month time points.

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Figure 6. Alveolar volume. The number-weighted alveolar volume in the right middle lung lobe for four treatment groups at 3 and 6 months is represented. There was a significant increase (*) (P ≤ 0.05) between the O3 and O3 + HDMA compared to the FA groups at 3 months of age. From 3 to 6 months the alveolar volume decreased in the O3 + HDMA only (**) (P ≤ 0.05).

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Figure 7. Distribution of alveolar volume. The coefficient of variation of the distribution of number-weighted alveolar volumes (CVn alv) in the right middle lung lobe for four treatment groups at 3 and 6 months is represented. There was a significant decrease (*) (P ≤ 0.05) between the O3 and O3 + HDMA compared to the FA groups at 3 and 6 months of age.

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Alveolar Capillary Surface Density

Both age and exposure influence changes in alveolar capillary surface density (Table 3). Figure 8 displays the mean alveolar capillary surface density represented as the surface density of alveolar capillaries per IAS. The surface density is significantly less for the ozone groups (ozone and HDMA/ozone) compared to the FA groups in the 3-month group. There is no difference in capillary surface density between exposure groups at the 6-month time point. In addition, there is a significant decrease in capillary surface density from 3 to 6 months within the FA group. Across all age groups, ozone had a greater effect on capillary surface density than did HDMA (Table 3). These results demonstrate that during normal alveolar growth in the early postnatal period, capillary surface density decreases from 3 to 6 months. Monkeys exposed to ozone have an alveolar capillary surface density that is unchanged from 3 to 6months. This would imply that there is a normal biological process to decrease capillary surface density from 3 to 6 months that does not occur in ozone exposed monkeys or occurs earlier in the postnatal period within the ozone exposure monkeys.

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Figure 8. Capillary surface density. The ratio of the pulmonary capillary to interalveolar septal surface (Ss cap, IAS) in the right middle lung lobe for four treatment groups at 3 and 6 months is represented. There was a significant decrease (*) (P ≤ 0.05) between the O3and O3 + HDMA compared to the FA groups at 3 months. From 3 to 6 months there was a significant decrease in capillary surface density in the FA and HDMA groups (**) (P ≤ 0.05) and a significant increase within the O3 group. (†) (P ≤ 0.05).

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Changes in Relative Gene Expression

We used a candidate gene approach to evaluate relative changes in the expression of genes related to angiogenesis and alveolar growth. Using only lung parenchyma, relative gene expression was quantified for each rhesus macaque at the 3- and 6-month group. Relative differences between treatment groups at either 3 months of age or 6 months of age were not significant (data not shown). As the most significant changes in alveolar number, size, and capillary surface density occurred over time, we analyzed the relative gene expression changes for each gene over time. FA monkeys served as the comparison control, where the expression of each gene was represented as a change from its 3-month value. The relative difference of each gene between 6 and 3 months for each of the exposure groups (HDMA, ozone, ozone +HDMA) was then compared to the relative difference for each gene in the FA group. The results are summarized in Table 4. Compared to the FA group, TGFβ expression was decreased, most significantly in the combined ozone/HDMA group. Conversely, the ozone group demonstrated a significant increase in keratinocyte growth factor and acidic fibroblast growth factor gene expression compared to the FA group, which demonstrated a decrease in the expression of these two growth factors from 3 to 6 months. In addition, the expression of two anti-angiogenic factors, pigment epithelial derived growth factor (PEDF), and placental growth factor (PlGF) demonstrated a trend toward an increase in gene expression in the HDMA exposed group. Relative expression of PlGF was decreased from 3 to 6 months in the ozone exposed groups and increased in the FA and HDMA groups.

Table 4. Summary of gene expression changes between 3 and 6 months of age (statistically significant changes compared to Filtered Air controls are highlighted in bold)
GeneFA (ΔΔCt)HD (ΔΔCt)P valueOzone (ΔΔCt)P valueHD/Ozone (ΔΔCt)P value
V701.37000.994NS0.8790NS1.2500NS
V550.99200.6499NS0.9970NS0.8730NS
P3110.57401.2100NS1.1090NS0.9890NS
TGFβ1.18000.75200.02200.85200.06000.57500.0030
HGF0.96601.3430NS1.3640NS1.1610NS
KGF0.87901.07000.05101.32200.00201.0060NS
VEGFr11.59001.5600NS2.0620NS1.1530NS
VEGFr21.10001.1000NS1.1500NS0.7800NS
PDGFβ0.77401.0200NS1.1390NS0.7330NS
PDGFα0.84001.2880NS1.4740NS1.1800NS
PlGF1.62302.1100NS0.7800NS0.8960NS
PEDF0.90702.96600.00401.3050NS1.4810NS
Ang20.90101.5440NS1.5400NS1.4640NS
ANG11.23501.1900NS1.1800NS0.9640NS
bFGF0.64701.2200NS1.3600NS1.3730NS
aFGF0.88001.2600NS1.78300.0111.2330NS

DISCUSSION

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

The rhesus macaque has a very similar distribution and development of lung parenchyma to humans. As a result, it serves as an excellent model for studying early postnatal events in the development and growth of alveoli. The contribution of air pollution exposure and allergen exposure early in life to the development of chronic airway disease has previously been established, but the effect on alveolarization is currently not known (Miller et al., 2003). We do know that nonhuman primates are about fourfold more sensitive to inhaled ozone-induced injury than rodents making them the most appropriate model for humans (Plopper and Hyde, 2008). Recent epidemiological studies have shown that ozone exposure may increase hospital asthma admissions in young children, may be responsible for severe acute asthma attacks, and may be associated with lower lung function (Lin et al., 2008; Khatri et al., 2009). The contribution of allergen and ozone on alveolar growth and development has not been established.

We chose the rhesus macaque to study the effect of environmental exposure during the early postnatal period on the growth and development of alveoli. Rhesus Macaques at 1 month of age were separated into four separate exposure groups, FA, house dust mite antigen (HDMA), ozone (O3), and house dust mite combined with ozone (HDMA/O3). One cohort was taken to necropsy at 3 months while a second cohort was taken to necropsy at 6 months. The FA group served as the control group and represented normal development. The lung volume, number and volume of alveoli, and capillary surface density per alveolar septa were determined using previously described methods (Hyde et al., 2004). Quantitative gene expression analysis of candidate genes was performed using only the lung parenchyma devoid of airways.

Previous work, predominantly in rodents, has established that the number of alveoli increases from thousands at birth to several hundred million by adulthood (Ochs et al., 2004). Traditional dogma states that the initially immature alveoli, characterized by a double capillary network, undergo microvascular maturation where capillary surface density is decreased by half. This stage is followed by the slow addition of more alveoli commonly termed late alveolarization (Burri, 2006). Studies have suggested that late alveolarization still involves capillary upfolding from immature septa in the adult lung or from the pleura or perivascular cuffs (Burri, 2006). Studies in rodents have identified that approximately 40% of alveoli are added after microvascular maturation, arising from mature single capillary septa (Schittny et al., 2008). There are morphometric differences between the rodent lung and primate lung, raising the possibility that the previously described patterns that are seen on rodents may not apply to human alveolar development (Plopper and Hyde, 2008). This study is the first to examine the effect of a stress response on alveolar development in a nonhuman primate model.

The patterns of normal development are represented in the by patterns of the FA group from the 3- to 6-month time point. The lung volume and alveolar number proportionally increased from 3 to 6 months while the distribution of size of alveoli was fairly wide. The increased number of alveoli was proportional to the increase in lung volume, consistent with prior observations (Hyde et al., 2007). The size, or volume, of alveoli changed insignificantly from 3- to 6 months. By contrast, the density of capillaries per septa was decreased by 50% from 3 to 6 months. The HDMA group had a much more significant increase in lung volume and alveolar number compared to the FA group while also having a fairly wide distribution of alveolar size. The HDMA group also had a decrease in capillary surface density from 3 to 6 months.

The Ozone and Ozone/HDMA combined groups began with fewer but larger alveoli than that of the FA groups. At the 6-month time point, the ozone and ozone/HDMA had an equal number of alveoli as the FA and HDMA exposure groups. The size of alveoli remained larger for the Ozone and Ozone/HDMA groups at both 3 and 6 months. This “catching up” in number of alveoli was characterized by a fairly uniform increase with respect to distribution of size. The density of capillaries per alveolar septae remained the same for the Ozone/HDMA group and very slightly increased for the Ozone group from 3 to 6 months in contrast to the large decrease in capillary surface density in the FA and HDMA groups. Therefore, the highly significant increase in alveolar number within the 2 Ozone groups occurred without any changes in capillary surface density. This “late alveolarization” occurred with minimal change in microvascular growth and development. The alveoli were structurally larger than the alveoli of the FA groups resulting in a larger lung volume. Isolating ozone effects from HDMA effects, the changes described above were primarily seen in the ozone exposed groups. Significant loss of IAS has been observed following exposure to ambient levels of other inhaled oxidants, NO2, and NO (1.21 and 0.31 mg/m3) for 16 hr/day for 68 months, followed by 36 months recovery in FA in beagle dogs (Hyde et al., 1978; Harkema et al., 1993). The lesions observed in chronically exposed dogs were marked by multifocal centriacinar airway enlargement and some interalveolar septal destruction. This study in beagle dogs raises two issues for our current study in rhesus monkeys that, (1) the period of exposure may not have been long enough to observe the full effect of ozone inhalation and (2) the postexposure period may have different mechanisms of injury and repair than during the exposure period. However, the current study in rhesus macaques does identify that alveolar number can be affected by early life exposure to ozone and if the exposure continues it may lead to a more permanent loss of alveoli (Harkema et al., 1993). We used a candidate gene expression based approach to try and identify phenotypically silent microvascular development. We chose genes that were previously established to play a role in angiogenesis and alveolar maturation. Relative gene expression analysis confirmed an active gene expression program of microvascular maturation in the FA and HDMA groups. The rise in antiangiogenic PEDF and PlGF correlated with a decrease in microvascular density in the FA and HDMA groups. In contrast, genes associated with mesenchymal growth, KGF and FGF, were increased in the ozone exposed groups. Both KGF and FGF have been described in previous work as promoting postnatal alveolar formation(Kaza et al., 2002; Morris et al., 2003; Srisuma et al., 2010; Yildirim et al., 2010).TGFβ expression was lower in all of the exposure groups compared FA. A decrease in TGFβ has previously been described as playing a role in septation and development of emphysema (Morris et al., 2003; Yildirim et al., 2010). Interestingly, there was a trend toward an increase in p311 expression in all three of the exposure groups as well, whereas the FA group trended toward a decrease in p311. These results were not significant but are interesting because p311 has been shown to bind latent TGFβ and preventing conversion of TGFβ toward the active form (Paliwal et al., 2004). Most significantly, there was no evidence of increased gene expression of any major angiogenic factor in the group exposed to ozone.

This study has established that ozone exposure during the early postnatal period alters normal alveolar development. Ozone exposed rhesus macaque start with a smaller number of larger alveoli, which increase in number without a microvascular maturation phase. The addition of alveoli during this late stage of development is given the term “late alveolarization.” The mechanism of this late alveolarization is not clear but may involve promotion of epithelial mesenchymal maturation and elastogenesis. The consequences of these programmatic changes in alveolarization may have significant consequences for future disease and altered lung capacity during the adult years. Indeed, there has been greater attention on the phenotypes of asthma, with recognition that progressive and persistent disease may be a unique “cluster” of patients (Sorkness et al., 2008). This same “cluster” of persistent impairment of lung function associated with poor clinical outcomes has also been recognized in children (Fitzpatrick et al., 2011). Asthmatics with a poor bronchodilator response, “air-trapping”, and decline in lung function may have a parenchymal impairment that includes a decrease in alveolar number. Phenotypically, this clinical cluster of severe asthma may share pathophysiology with COPD including alteration of alveolar injury and repair mechanisms. The relationship between allergen and ozone exposure and severity of asthma and allergy symptoms has long been recognized (Delfino et al., 1996; Kim et al., 2011). Having a better understanding of the molecular process responsible for the early alveolar loss and subsequent increase in alveolar growth and the influence of ozone and allergens on this process may help identify key biomarkers and molecular targets for disease prevention and disease modifying therapy. Although the current model did not provide a specific mechanism for late alveolarization, it does represent an excellent model for studying alveolar injury and repair.

Acknowledgements

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

The authors thank Laurie Brignolo, D.V.M., Sarah Davis, and Bruce Rodello of the Cailfornia National Primate Research Center for their clinical and organizing expertise. The authors also thank Brian Tarkington, Jessica Arturus, Collette Brown, Carmen Ip, and Amanda Omlar for their technical assistance in data collection.

LITERATURE CITED

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