The subsequent biological effects of simulated microgravity on endothelial cell growth in HUVECs

Abstract

Purpose: Microgravity is known to cause endothelium dysfunction in astronauts returning from spaceflight. We aimed to reveal the regulatory mechanism in alterations of human endothelial cells after simulated microgravity (SMG).

Methods: We utilized the rotary cell culture system (RCCS-1) to explore the subsequent effects of SMG on human umbilical vein endothelial cells (HUVECs).

Results: SMG-treated HUVECs appeared obvious growth inhibition after return to normal gravity, which might be attributed to a set of responses including alteration of cytoskeleton, decreased cell adhesion capacity and increased apoptosis. Expression levels of mTOR and its downstream Apaf-1 were increased during subsequent culturing after SMG. miR-22 was up-regulated and its target genes SRF and LAMC1 were down-regulated at mRNA levels. LAMC1 siRNAs reduced cell adhesion rate and inhibited stress fiber formation while SRF siRNAs caused apoptosis.

Conclusion: SMG has the subsequent biological effects on HUVECs, resulting in growth inhibition through mTOR signaling and miR-22-mediated mechanism.

Keywords: Growth inhibition; LAMC1; Serum response factor; Simulated microgravity; mTOR; miR-22.

Fig. 1

Cellular changes during subsequent culturing after SMG treatment. A: Cell growth curve. B: Cell adhesion rate. C: Actin stress fibers were stained with the Rhodamine-conjugated phalloidin. Scale bar, 25 μm. D: Number of actin stress fibers was presented. *p < 0.05, **p < 0.01 vs G. Data was presented at indicated time points in the G and SMG groups after return to normal gravity. R indicates return to normal gravity.

Fig. 2

The subsequent effect of SMG on apoptosis of HUVECs. Flow cytometry analysis was performed as described in Materials and methods. A: Early and later apoptotic rates were shown in representative histograms. B: Percentage of total apoptotic cells was presented in different groups. C: The relative expression level of mTOR mRNA was quantified by qRT-PCR analysis. D: Apaf-1 protein was examined by western blot analysis. *p < 0.05. **p < 0.01 vs G. R indicates return to normal gravity.

Fig. 3

The subsequent effect of SMG on expression levels of miR-22 and its target genes in HUVECs. A: Relative expression level of miR-22 was shown in SMG group compared to G group. B: SRF and LAMC1 at mRNA levels were quantified by qRT-PCR analysis in SMG group compared to G group at 72 h after return to normal gravity. *p < 0.05 vs G. C: The 3′-UTR fragments of human SRF mRNA contain three putative miR-22 binding sites and mutant sequence (CGTCGA) was designed. D: pmiR-SRF-WT or pmiR-SRF-Mut was cotransfected with miR-22 mimics or miR-NC for 48 h. Luciferase activity was presented by relative hRlu/hluc ratio. **p < 0.01 vs miR-NC.

Fig. 4

The effects of SRF siRNAs on cell growth and apoptosis in HUVECs. A: The expression level of SRF protein was examined by western blot analysis. B: Cell numbers were counted in different groups. C: Early and later apoptotic rates were shown representative histograms. D: Percentage of total apoptotic cells was presented in different groups. *p < 0.05 vs NC siRNA.

Fig. 5

LAMC1 is a direct target of miR-22. A: The 3′-UTR fragments of human LAMC1 mRNA contain three putative miR-22 binding sites. Mut1 is deletion of seed region and Mut2 is base substitution in seed region. B: pmiR-LAMC1-WT or pmiR-LAMC1-Mut1/2 was cotransfected with miR-22 mimics or miR-NC for 48 h. Luciferase activity was presented by relative hRlu/hluc ratio. **p < 0.01 vs miR-NC.

Fig. 6

The effects of LAMC1 siRNAs on cell adhesion and actin cytoskeleton in HUVECs. A: The expression level of LAMC1 protein was examined by western blot analysis. B: Cell adhesion rate were analyzed in different groups at 4h after subculture. C: HUVECs were stained with the Rhodamine-conjugated phalloidin (red) and FAK (green) at 48 h after transfection with NC siRNA or LAMC1 si-1. The representative images show alteration of actin cytoskeleton. Scale bar, 25 μm *p < 0.05, **p < 0.01 vs NC siRNA.