An age-wise comparison of human airway smooth muscle proliferative capacity

Abstract

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca2+]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.

Fig 1. Proliferation of neonatal and adult cultured human airway smooth muscle cells in the presence of FCS and PDGF-AA.

A. Cell counts according to time. Results are means ± SEM. In glucose + 10% FCS medium, ASMC proliferation was greater in neonatal cells (open symbols, n = 5) vs. adult cells (closed symbols, n = 5). *p < 0.05 neonate vs. adult at the corresponding time using MANOVA. B. DNA synthesis in adult and neonatal cultured human airway smooth muscle cells. Results are means ± SEM. Values were normalized to proliferation in ITS medium. Cells cultured in 10% FCS (white bars, neonates, n = 5; black bars, adults, n = 7)), 15 ng/ml PDGF-AA (neonate, n = 4; adults, n = 10). *p < 0.05 using Mann-Whitney U-test.

Fig 2. Proliferation of neonatal and adlult cultured human airway smooth muscle cells in the presence of pro-inflammatory stimuli.

Results are means ± SEM. Values were normalized to proliferation in ITS medium. Cells cultured in 100 ng/ml IL-4 (black bars, adults; white bars, neonates)), IL-6, TNFalpha or 10^-4M Histamine, SLIGKV were assayed 24 following synchronization in ITS medium. There were no significant difference between the 2 age groups regarding all experimental conditions (N = 3 to 6 per group, Mann-Whitney U-test).

Fig 3. Porin expression according to age groups.

A. Immunoblot for porin and actin in neonatal and adult ASMC. B. Expression of porin in human cultured ASMC. A significant difference between the two cell populations after incubation in ITS medium for 1 day was found (neonates, white bars, n = 4 and adults, black bars, n = 3, *p < 0.05 neonate vs. adult using Mann-Whitney U-test.

Fig 4. Effect of mitochondrial metabolism (aerobic glycolysis) on ASMC proliferation.

A. Cell count according to the presence of glucose or not (galactose) in the culture medium. Results are means ± SEM. In galactose + 10% FCS medium, proliferation of neonatal ASMC (open circles, n = 6) is still present, unlike in adult ASMC (closed circles, n = 4). *p < 0.05 neonate vs. adult at the corresponding time using MANOVA. B. DNA synthesis according to the presence or absence of glucose in the culture medium. Values are means ± SEM. After 24h in a glucose-free (galactose) medium, neonatal ASMC (white bars, n = 5), DNA synthesis is still present, in contrast with adult ASMC (black bars, n = 3). *p < 0.05 using Mann-Whitney U-test.