Yong Gao

Periostin downregulation is an early marker of inhibited neonatal murine lung alveolar septation

BACKGROUND Extreme preterm birth exposes the saccular lung to multiple teratogens, which ultimately retard alveolar development. Specifically, therapeutic high level oxygen supplementation adversely affects the premature lungs and results in blunted alveolarization. Prolonged hyperoxic lung injury has previously been shown to upregulate the matricellular protein Periostin (Postn) and stimulate ectopic accumulation of alpha smooth muscle actin (αSMA) myofibroblasts. Therapies that promote lung septation are lacking largely due to a lack of reliable early biomarkers of injury. Thus, we determined if Postn expression correlated with the initial appearance of myofibroblasts in the saccular lung and was required for early alveolar development. METHODS Lung development in C57BL/6J mice following room-air (RA, 21%-O₂) or continuous hyperoxia (85%-O₂) from birth (P0) through postnatal day P14 was correlated with Postn and αSMA expression. Alveolarization in Postn knockout mice exposed to room-air, 60%-, and 85%-O₂ was also examined. RESULTS Postn was widely expressed in distal lung septa through P2 to P4 and peak expression coincided with accumulation of saccular myofibroblasts. Initially, 85%-O₂ prematurely downregulated Postn and αSMA expression and suppressed proliferation before the first evidence of distal lung simplification at P4. By P14, chronic 85%-O₂ resulted in secondary upregulation of Postn and αSMA in blunted septa. Myofibroblast differentiation and alveolar development was unaffected in Postn null mice and acute 85%-O₂ exposure equally inhibited septal formation in Postn null and wild-type littermates. CONCLUSION Postn expression is tightly correlated with the presence of αSMA-myofibroblasts and is a novel early biomarker of acutely inhibited alveolar septation during a crucial window of lung development.

Initial Suppression of Transforming Growth Factor-β Signaling and Loss of TGFBI Causes Early Alveolar Structural Defects Resulting in Bronchopulmonary Dysplasia

Septation of the gas-exchange saccules of the morphologically immature mouse lung requires regulated timing, spatial direction, and dosage of transforming growth factor (TGF)-β signaling. We found that neonatal hyperoxia acutely initially diminished saccular TGF-β signaling coincident with alveolar simplification. However, sustained hyperoxia resulted in a biphasic response and subsequent up-regulation of TGF-β signaling, ultimately resulting in bronchopulmonary dysplasia. Significantly, we found that the TGF-β-induced matricellular protein (TGFBI) was similarly biphasically altered in response to hyperoxia. Moreover, genetic ablation revealed that TGFBI was required for normal alveolar structure and function. Although the phenotype was not neonatal lethal, Tgfbi-deficient lungs were morphologically abnormal. Mutant septal tips were stunted, lacked elastin-positive tips, exhibited reduced proliferation, and contained abnormally persistent alveolar α-smooth muscle actin myofibroblasts. In addition, Tgfbi-deficient lungs misexpressed TGF-β-responsive follistatin and serpine 1, and transiently suppressed myofibroblast platelet-derived growth factor α differentiation marker. Finally, despite normal lung volume, Tgfbi-null lungs displayed diminished elastic recoil and gas exchange efficiency. Combined, these data demonstrate that initial suppression of the TGF-β signaling apparatus, as well as loss of key TGF-β effectors (like TGFBI), underlies early alveolar structural defects, as well as long-lasting functional deficits routinely observed in chronic lung disease of infancy patients. These studies underline the complex (and often contradictory) role of TGF-β and indicate a need to design studies to associate alterations with initial appearance of phenotypical changes suggestive of bronchopulmonary dysplasia.

Neonatal hyperoxic lung injury favorably alters adult right ventricular remodeling response to chronic hypoxia exposure.

The development of pulmonary hypertension (PH) requires multiple pulmonary vascular insults, yet the role of early oxygen therapy as an initial pulmonary vascular insult remains poorly defined. Here, we employ a two-hit model of PH, utilizing postnatal hyperoxia followed by adult hypoxia exposure, to evaluate the role of early hyperoxic lung injury in the development of later PH. Sprague-Dawley pups were exposed to 90% oxygen during postnatal days 0-4 or 0-10 or to room air. All pups were then allowed to mature in room air. At 10 wk of age, a subset of rats from each group was exposed to 2 wk of hypoxia (Patm = 362 mmHg). Physiological, structural, and biochemical endpoints were assessed at 12 wk. Prolonged (10 days) postnatal hyperoxia was independently associated with elevated right ventricular (RV) systolic pressure, which worsened after hypoxia exposure later in life. These findings were only partially explained by decreases in lung microvascular density. Surprisingly, postnatal hyperoxia resulted in robust RV hypertrophy and more preserved RV function and exercise capacity following adult hypoxia compared with nonhyperoxic rats. Biochemically, RVs from animals exposed to postnatal hyperoxia and adult hypoxia demonstrated increased capillarization and a switch to a fetal gene pattern, suggesting an RV more adept to handle adult hypoxia following postnatal hyperoxia exposure. We concluded that, despite negative impacts on pulmonary artery pressures, postnatal hyperoxia exposure may render a more adaptive RV phenotype to tolerate late pulmonary vascular insults.

Relationship of Structural to Functional Impairment during Alveolar-Capillary Membrane Development

Bronchopulmonary dysplasia is a chronic lung disease of extreme preterm infants and results in impaired gas exchange. Although bronchopulmonary dysplasia is characterized histologically by alveolar-capillary simplification in animal models, it is clinically defined by impaired gas diffusion. With the use of a developmentally relevant model, we correlated alveolar-capillary structural simplification with reduced functional gas exchange as measured by the diffusing factor for carbon monoxide (DFCO). Neonatal mouse pups were exposed to >90% hyperoxia or room air during postnatal days 0 to 7, and then all pups were returned to room air from days 7 to 56. At day 56, DFCO was measured as the ratio of carbon monoxide uptake to neon dilution, and lungs were fixed for histologic assessment of alveolar-capillary development. Neonatal hyperoxia exposure inhibited alveolar-capillary septal development as evidenced by significantly increased mean linear intercept, increased airspace-to-septal ratio, decreased nodal density, and decreased pulmonary microvasculature. Importantly, alveolar-capillary structural deficits in hyperoxia-exposed pups were accompanied by a significant 28% decrease in DFCO (0.555 versus 0.400; P < 0.0001). In addition, DFCO was highly and significantly correlated with structural measures of reduced alveolar-capillary growth. Simplification of alveolar-capillary structure is highly correlated with impaired gas exchange function. Current mechanistic and therapeutic animal models of inhibited alveolar development may benefit from application of DFCO as an alternative physiologic indicator of alveolar-capillary development.

Cumulative effects of neonatal hyperoxia on murine alveolar structure and function

Bronchopulmonary dysplasia (BPD) results from alveolar simplification and abnormal development of alveolar and capillary structure. Survivors of BPD display persistent deficits in airflow and membrane and vascular components of alveolar gas diffusion. Despite being the defining feature of BPD, various neonatal hyperoxia models of BPD have not routinely assessed pulmonary gas diffusion.