john torday

ABSTRACT Although alveolar wall thinning has been attributed to apoptosis of interstitial lung lipofibroblasts (LFs), the underlying molecular mechanism(s) remains unknown. Although the physiological vitamin D steroid hormone 1alpha,25(OH)(2)D(3) (1,25D) has been suggested as a local paracrine/autocrine effector of fetal lung maturation and is known to affect fibroblast apoptosis, its effects on LF apoptosis are unknown. We determined the role of 1,25D and its metabolite, C-3-epimer (3-epi-1,25D), on LF and alveolar type II (ATII) cell differentiation, proliferation, and apoptosis. Embryonic day 19 Sprague-Dawley fetal rat lung LFs and ATII cells were treated with 1,25D or 3-epi-1,25D (1 x 10(-10) to 1 x 10(-8) M) for 24 h, and cell proliferation, apoptosis, and differentiation were assessed. Both 1,25D and 3-epi-1,25D exhibited dose-dependent increases in expression of the key homeostatic epithelial-mesenchymal differentiation markers, increased LF and ATII cell proliferation, and decreased apoptosis. Furthermore, rat pups administered 1,25D from postnatal days 0 to 14 showed increased expressions of key LF and ATII cell differentiation markers, increased Bcl-2-to-Bax ratio as an index of decreased spontaneous alveolar LF and ATII cell apoptosis, increased alveolar count, and a paradoxical increase in septal thickness. We conclude that spatial- and temporal-specific actions of vitamin D play a critical role in perinatal lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and by modulating LF proliferation/apoptosis. These data not only provide the biological rationale for the presence of an alveolar vitamin D paracrine system, but also provide the first integrated molecular mechanism for increased surfactant synthesis and alveolar septal thinning during perinatal lung maturation.

Functional maturation of pulmonary alveolar epithelial cells is crucial for extrauterine survival. Mechanical distension and mesenchymal-epithelial interactions play important roles in this process. We hypothesized that mechanical stretch simulating fetal breathing movements is an important regulator of pulmonary epithelial cell differentiation. Using a Flexercell Strain Unit, we analyzed effects of stretch on primary cultures of type II cells and cocultures of epithelial and mesenchymal cells isolated from fetal rat lungs during late development. Cyclic stretch of isolated type II cells increased surfactant protein (SP) C mRNA expression by 150 +/- 30% over controls (P < 0.02) on gestational day 18 and by 130 +/- 30% on day 19 (P < 0.03). Stretch of cocultures with fibroblasts increased SP-C expression on days 18 and 19 by 170 +/- 40 and 270 +/- 40%, respectively, compared with unstretched cocultures. On day 19, stretch of isolated type II cells increased SP-B mRNA expression by 50% (P < 0.003). Unlike SP-C, addition of fibroblasts did not produce significant additional effects on SP-B mRNA levels. Under these conditions, we observed only modest increases in cellular immunoreactive SP-B, but secreted saturated phosphatidylcholine rose by 40% (P < 0.002). These results indicate that cyclic stretch promotes developmentally timed differentiation of fetal type II cells, as a direct effect on epithelial cell function and via mesenchymal-epithelial interactions. Expression of the SP-C gene appears to be highly responsive to mechanical stimulation.

AbstractThe First Principles for Physiology originated in and emanate from the unicellular state of life. Viewing physiology as a continuum from unicellular to multicellular organisms provides fundamental insight to ontogeny and phylogeny as a functionally integral whole. Such mechanisms are most evident under conditions of physiologic stress; all of the molecular pathways that evolved in service to the vertebrate water–land transition aided and abetted the evolution of the vertebrate lung, for example. Reduction of evolution to cell biology has an important scientific feature—it is predictive. One implication of this perspective on evolution is the likelihood that it is the unicellular state that is actually the object of selection. By looking at the process of evolution from its unicellular origins, the causal relationships between genotype and phenotype are revealed, as are many other aspects of physiology and medicine that have remained anecdotal and counter-intuitive. Evolutionary development can best be considered as a cyclical, epigenetic, reiterative environmental assessment process, originating from the unicellular state, both forward and backward, to sustain and perpetuate unicellular homeostasis. WIREs Syst Biol Med 2015, 7:275–284. doi: 10.1002/wsbm.1305For further resources related to this article, please visit the WIREs website.

The synthesis of proteoglycans by primary cultures of 19-day gestation fetal rat lung Type II pneumonocytes was studied. The cells were grown in the presence of [3H]-glucosamine and/or [35S]-Na2SO4 and the radioactive label incorporated into proteoglycans was analyzed. Proteoglycans of high molecular weight (approximately 200 Kd) were isolated by gel permeation chromatography and contained both [3H] and [35S]. The glycosaminoglycan composition of the proteoglycans was determined by electrophoresis and autoradiography. The medium contained 65-80% of the labeled proteoglycans and was enriched for hyaluronate, with lesser amounts of the sulfated glycosaminoglycans (dermatan sulfate greater than heparan sulfate greater than chondroitin sulfate). The cell layers retained 20-35% of the labeled proteoglycans and was enriched for heparan sulfate, with lesser amounts of chondroitan sulfate greater than dermatan sulfate greater than hyaluronate. The synthesis of proteoglycans was time-dependent and was stimulated by increasing concentrations of fetal bovine serum. Cortisol inhibited proteoglycan synthesis, apparently by decreasing the availability of proteoglycan core-protein.

Acknowledgments: This study was ,supported ,by grants ,from ,NIH (HL55268 and HL075405), AHA (10868-02), Philip Morris USA, Inc. and Philip Morris International (11108-02) and TRDRP (14RT-0073 and 15IT-0250). Running title-Mechanism for the paradoxical ………. Address for Reprint Requests and Correspondence: Virender K. Rehan, MD Department,of Pediatrics Los Angeles Biomedical,Research Institute at Harbor UCLA Medical Center David Geffen School of Medicine

Fetal breathing movements (FBM) are necessary for fetal lung growth and maturation. The authors analyzed fetal rat lungs cultured with or without lung distension and tracheal ligation, and examined the effects of mechanical stretch on a human pulmonary epithelial cell line (NCI-H441) that shows regulated expression of surfactant proteins (SP-A, SP-B). Cells were grown on silastic membranes and mounted in a Flexercell Strain Unit. Cyclic deformation simulating FBM was achieved by applying a vacuum of 22 kPa (5%-15% radial deformation) at 50 cycles per minute for 2 to 24 hours. Results indicate that static distension for as little as 4 hours decreased steady-state SP-A and SP-B mRNA levels in whole lung (n = 5-6, P < .01). In contrast, cyclic stretching of H441 cells for 24 hours increased SP-B and SP-A expression 2- to 4-fold over controls. Cyclic deformation also significantly enhanced 3H-choline incorporation into saturated phosphatidylcholine. Dynamic mechanodeformation may be a critical stimulus for fetal lung development.

The underlying mechanisms and effector molecules involved in mediating in utero smoke exposure-induced effects on the developing lung are only beginning to be understood. However, the effects of a newly discovered category of smoke, i.e., thirdhand smoke (THS), on the developing lung are completely unknown. We hypothesized that, in addition to nicotine, other components of THS would also affect lung development adversely. Fetal rat lung explants were exposed to nicotine, 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the two main tobacco-specific N-nitrosamine constituents of THS, for 24 h. We then determined key markers for alveolar paracrine signaling [epithelial differentiation markers surfactant phospholipid and protein synthesis; mesenchymal differentiation markers peroxisome proliferator-activated receptor γ (PPAR-γ), fibronectin and calponin], the BCL-2-to-Bax ratio (BCL-2/Bax), a marker of apoptosis and the involvement of nicotinic acetylcholine receptors (nAChR)-α3 and -α7 in mediating NNA's and NNK's effects on the developing lung. Similar to the effects of nicotine, exposure of the developing lung to either NNK or NNA resulted in disrupted homeostatic signaling, indicated by the downregulation of PPAR-γ, upregulation of fibronectin and calponin protein levels, decreased BCL-2/Bax, and the accompanying compensatory stimulation of surfactant phospholipid and protein synthesis. Furthermore, nAChR-α3 and -α7 had differential complex roles in mediating these effects. NNK and NNA exposure resulted in breakdown of alveolar epithelial-mesenchymal cross-talk, reflecting lipofibroblast-to-myofibroblast transdifferentiation, suggesting THS constituents as possible novel contributors to in utero smoke exposure-induced pulmonary damage. These data are particularly relevant for designing specific therapeutic strategies, and for formulating public health policies to minimize THS exposure.

Obesity and its related diseases are the leading cause of death in western society, with associated risks of hypertension, coronary heart disease, stroke, diabetes, and breast, prostate and colon cancer. Recent epidemiologic data indicate an increased risk of Alzheimer's disease in association with adult obesity. There is now convincing evidence that, in both human and animal models, the in utero environment may impact on fetal developmental processes, altering offspring homeostatic regulatory mechanisms. "Gestational programming" may result in altered cell number, organ structure, hormonal set points or gene expression, with effects being permanent or expressed only at select offspring ages (e.g., newborn, adult). Our laboratory and others have demonstrated that low birth weight rats, induced by maternal food restriction or uterine artery ligation, paradoxically develop adult obesity with glucose intolerance and hypertension. Recent studies indicate alterations in peripheral (hepatic) and central (hippocampus) IGF-1 gene expression and epigenetic regulation among these offspring. These findings suggest that potential risk factors for the development of Alzheimer's disease may be present as early as newborn life.

Alveolar type II cells that were isolated from organotypic cultures of fetal rat lung and maintained in primary monolayer culture were analyzed. Morphologically the cells retained many characteristics of type II cells in vivo. Biochemical analysis revealed that the cells contained and synthesized phosphatidylcholine with a high degree of saturation. Furthermore, the cells released saturated phosphatidylcholine and responded to adrenergic stimulation with increased release. The type II cells retained their ability to proliferate and responded to fibroblast-pneumonocyte factor. These data indicate that primary cultures of fetal type II cells will be useful to elucidate mechanisms modulating type II cell proliferation, surfactant synthesis, and surfactant secretion.

Phosphatidylcholine (PC) synthesis in cultured fetal rabbit lung cells was studied after exposure to various pituitary peptides. Because of reports of an association between lung maturation and prolactin, 2 different preparations of ovine prolactin were studied in concentrations ranging from 0.1 to 10.0 microgram/ml. Increased saturated phosphatidylcholine (SPC) synthesis without an increase in synthesis of total PC, and increased SPC/PC, was observed after exposure to Sigma prolactin. NIH prolactin in the same concentrations had no effect on synthesis of either PC or SPC. Adrenocorticotropic hormone, luteinizing hormone and thyroid stimulating hormone increased synthesis of both PC and SPC, but none increased the SPC/PC ratio. A cold ethanol extraction of Sigma prolactin yielded fractions with no demonstrable biologic activity, and other initial attempts to characterize the active factor in the Sigma prolactin samples were unsuccessful. We concluded that prolactin per se has no direct effect on PC or SPC synthesis in the lung, but that certain other pituitary peptides have a direct effect on lung synthesis of PC and SPC.

The mature lung produces pulmonary surfactant, a lipid-protein complex that prevents lung alveoli from becoming atelectatic. The prenatal surge of surfactant production in preparation for birth occurs between 28-34 weeks gestation and is triggered by glucocorticoids, which stimulate surfactant synthesis by alveolar epithelial cells through a series of biochemical steps mediated by mesenchymal-epithelial interactions. In contrast to this, when explanted midgestation human fetal lung tissue is maintained in serum-free medium, there is a spontaneous increase (40%) in de novo saturated phosphatidylcholine (SPC) synthesis on the fifth day in culture. Addition of dexamethasone (DEX; 1 x 10(-8) M) to the culture medium causes the increased synthesis in SPC to occur earlier (day 4) and to a greater extent (87%). Addition of an equimolar concentration of dihydrotestosterone (DHT) to the medium delays both the spontaneous and DEX-stimulated increases in SPC synthesis by 24 h. Weak fetal adrenal androgens are also able to block both spontaneous and DEX-stimulated SPC synthesis. Addition of DHT to the explant cultures at daily intervals reveals that inhibition of the DEX effect occurs within the first 24 h after exposure. The antiandrogen flutamide neutralizes the effect of DHT, indicating that it acts through the androgen receptor to block the glucocorticoid. Therefore, the human fetal adrenal cortex may time lung development through both inhibitory and stimulatory mechanisms.