Generation of a new immortalized human lung pericyte cell line: a promising tool for human lung pericyte studies

Abstract

Pericytes apposed to the capillary endothelium are known to stabilize and promote endothelial integrity. Recent studies indicate that lung pericytes play a prominent role in lung physiology, and they are involved in the development of various lung diseases including lung injury in sepsis, pulmonary fibrosis, asthma, and pulmonary hypertension. Accordingly, human lung pericyte studies are important for understanding the mechanistic basis of lung physiology and pathophysiology; however, human lung pericytes can only be cultured for a few passages and no immortalized human lung pericyte cell line has been established so far. Thus, our study aims to establish an immortalized human lung pericyte cell line. Developed using SV40 large T antigen lentivirus, immortalized pericytes exhibit stable SV40T expression, sustained proliferation, and have significantly higher telomerase activity compared to normal human lung pericytes. In addition, these cells retained pericyte characteristics, marked by similar morphology, and expression of pericyte cell surface markers such as PDGFRβ, NG2, CD44, CD146, CD90, and CD73. Furthermore, similar to that of primary pericytes, immortalized pericytes promoted endothelial cell tube formation and responded to different stimuli. Our previous data showed that friend leukemia virus integration 1 (Fli-1), a member of the ETS transcription factor family, is a key regulator that modulates inflammatory responses in mouse lung pericytes. We further demonstrated that Fli-1 regulates inflammatory responses in immortalized human lung pericytes. To summarize, we successfully established an immortalized human lung pericyte cell line, which serves as a promising tool for in vitro pericyte studies to understand human lung pericyte physiology and pathophysiology.

Figure 1.. Cell morphology and characteristics of normal and immortalized human lung pericytes.

(A) Normal and immortalized human lung pericyte morphology were assessed by phase contrast light microscopy. Scale bar: 50 μm. Pericytes were also assessed by immunostaining (B) and western blot (C-D) for PDGFRβ, neural glial antigen-2 (NG2) and SV40T. Nucleus stained blue and PDGFRβ or NG2 stained red. Magnification: 100x. HLPC: human lung pericytes.

Figure 2.. Relative telomerase activity of normal and immortalized human lung pericytes.

(A) Cumulative culture of normal and immortalized human lung pericytes. (B) Relative telomerase activity in normal and immortalized human lung pericytes at passage 9 were measured. N=3. *p < 0.05 compared to control human lung pericyte (HLPC) group.

Figure 3.. Representative histograms of cell-surface marker staining on immortalized human lung pericytes.

Immortalized human lung pericytes (P16) were stained for CD34 (A), CD45 (B), CD31 (C), CD44 (D), CD146 (E), CD90 (F), CD73 (G) and CD140b (H, PDGFR-β) and analyzed by flow cytometry.

Figure 4.. Response of normal and immortalized human lung pericytes to different stimuli.

Normal and immortalized human lung pericytes (HLPC) were stimulated with LPS (100 ng/ml), TNFα (10 ng/ml) or Thrombin (0.1 U) for 12h. (A-F) Total RNA were extracted and mRNA levels of IL-6, IL-8, VEGFα, MMP-9, CCL2 and G-CSF were measured by Real-time PCR. N=3 independent experiments. *p < 0.05 compared to control group.

Figure 5.. The effect of normal and immortalized human lung pericytes on endothelial cell tube formation under hypoxic conditions.

HUVECs were cultured under normal (21% oxygen) or hypoxic (1% oxygen) conditions without or with pericytes at 20:1 ratio (EC versus pericytes) in Matrigel-coated 24-well plate for 16h and then stained by Calcein AM fluorescent dye for 30 min. (A) Endothelial cell tube formation was observed and (B) the tube number was counted under a fluorescent microscope. Scale bar: 50 μm. N=3 independent experiments. *p < 0.05 compared to EC under 21% oxygen group. ^#p < 0.05 compared to EC under 1% oxygen group. EC: endothelial cell; NLPC: normal human lung pericytes; MLPC: immortalized human lung pericytes; HLPC: human lung pericytes.

Figure 6.. Fli-1 regulates inflammatory response in immortalized human lung pericytes.

Immortalized human lung pericytes were transfected with control or specific Fli-1 antisense gapmer for 24h and further stimulated with LPS for another 12 or 24h. mRNA (A) and protein (B) levels of IL-6 were measured. N=3 independent experiments. *p < 0.05 compared to control group. ^#p < 0.05 compared to LPS control group.