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. 2016 Jun 23;84(7):2141-2148.
doi: 10.1128/IAI.01305-15. Print 2016 Jul.

Endothelial Cell Response to Fusobacterium nucleatum

Reila Tainá Mendes  1   2 , Daniel Nguyen  1 , Danielle Stephens  1 , Ferda Pamuk  1   3 , Daniel Fernandes  2 , Thomas E Van Dyke  1 , Alpdogan Kantarci  4
Affiliations

Endothelial Cell Response to Fusobacterium nucleatum

Reila Tainá Mendes et al. Infect Immun. .

Abstract

Vascular response is an essential aspect of an effective immune response to periodontal disease pathogens, as new blood vessel formation contributes to wound healing and inflammation. Gaining a greater understanding of the factors that affect vascular response may then contribute to future breakthroughs in dental medicine. In this study, we have characterized the endothelial cell response to the common bacterium Fusobacterium nucleatum, an important bridging species that facilitates the activity of late colonizers of the dental biofilm. Endothelial cells were infected with Fusobacterium nucleatum (strain 25586) for periods of 4, 12, 24, or 48 h. Cell proliferation and tube formation were analyzed, and expression of adhesion molecules (CD31 and CD34) and vascular endothelial growth factor (VEGF) receptors 1 and 2 was measured by fluorescence-activated cell sorter (FACS) analysis. Data indicate that F. nucleatum impaired endothelial cell proliferation and tube formation. The findings suggest that the modified endothelial cell response acts as a mechanism promoting the pathogenic progression of periodontal diseases and may potentially suggest the involvement of periodontopathogens in systemic diseases associated with periodontal inflammation.

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Figures

FIG 1
FIG 1
HUVEC impairment of cell proliferation and tube formation. (A) F. nucleatum had no observable effect on endothelial cell proliferation at up to 4 h postinfection. After the 4-h mark, cell proliferation by infected endothelial cells occurred at a markedly lower rate than that observed in the control group. (B) Tube formation is impaired in cells infected with F. nucleatum in terms of both numbers of tubes and tube area; endothelial cells form tube-like structures after 12 h (control); endothelial cells infected with F. nucleatum for 24 h are unable to form tube-like structures (F. nucleatum). Magnification for images, ×400. (C) Average number of tubes (top), average area of tubes (middle), and number of cells per tube (bottom). *, P < 0.05.
FIG 2
FIG 2
F. nucleatum modulates expression of HUVEC surface markers. The percentage of endothelial cells expressing CD31 was seen to decrease after infection with F. nucleatum (A), while the percentage of endothelial cells expressing CD34 was seen to increase following infection (B). Measurements were taken at baseline levels and after 4, 12, and 24 h of infection with F. nucleatum, as analyzed by flow cytometry. *, P < 0.05 (compared to control results at 12 h); #, P < 0.05 (compared to control results at 4 h).
FIG 3
FIG 3
F. nucleatum modulates VEGF. (A and B) F. nucleatum was observed to modulate VEGFR1 (A) and VEGFR2 (B). Results are shown as percentages of expression of endothelial cells at baseline and following 4, 12, or 24 h of infection. (C and D) VEGF release in supernatants showed some fluctuation (C), while VEGF mRNA was downregulated (D) (data represent fold decreases after the 4- and 12-h marks expressed in a time-dependent manner). *, P < 0.05 (compared to control results at 4 h); #, P < 0.05 (compared to control results at 4 h and 12 h); **, P < 0.05 (compared 12 h results to control and 12 h results to 4 h).
FIG 4
FIG 4
F. nucleatum infection increases prostaglandin and cytokine release in supernatants and decreases mRNA PLCY1 expression in HUVEC. (A) The inflammatory component of supernatants is characterized by high-level release of PGE2 after 12 and 24 h. Endothelial cells infected with F. nucleatum were observed to release a high level of PGE2, suggesting a vasodilatory state. (B and C) Levels of IL-1α (B) and TNF-α (C) were increased after the 12- and 24-h marks following infection. (D) mRNA PLCY1 expression was reduced after the 4- and 12-h marks following infection (*, P < 0.05).
FIG 5
FIG 5
F. nucleatum modulates mRNA eNOS without changing mRNA iNOS or NOx release. (A) F. nucleatum infection was not observed to affect NOx release (measured as micromoles per liter) by endothelial cells at any time point. (B and C) After the 4-h mark following infection, levels of eNOS mRNA were observed to increase (B), while those of iNOS were not observed to be affected (C). (D) HIF-1α mRNA expression was analyzed by quantitative PCR (qPCR). Data show a reduction in HIF-1α expression after 4 and 12 h. *, P < 0.05 (compared to control results).
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