Differential responses of endothelial cells on three‐dimensional scaffolds to lipopolysaccharides from periodontopathogens

Abstract

Studies have been conducted on the pathogenicity of periodontopathogens in cultures of endothelial cells on two‐dimensional (2D) polystyrene surfaces, where the monolayer formed is not exposed to proteins of the subendothelial matrix. In this work, we developed a culture system by seeding human coronary artery endothelial cells (HCAECs) onto three‐dimensional (3D) scaffolds of collagen type I, a subendothelial protein. The inflammatory responses of the HCAEC monolayers, formed either on 3D scaffolds or directly on a 2D polystyrene plate, to lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) were evaluated. The transcription of 3 genes, the secretion of 40 cytokines and 2 prostanoids, and the adhesion of monocytes to 2D and 3D cultures with or without exposure to lipopolysaccharides (control) were assessed. HCAECs exhibited differences in transcriptional and secretory profiles between the 3D and 2D models. In addition, the inflammatory responses of HCAEC to Aa‐LPS and Pg‐LPS differed between the two models. In 3D cultures treated with Aa‐LPS, the levels of IL‐8, RANTES, G‐CSF, ICAM‐1, IL‐6, and TXA2 were significantly higher than those in the controls. In 2D cultures treated with Aa‐LPS, IL‐8, RANTES, G‐CSF, ICAM‐1, TNF‐RI, PGI2, and TXA2 levels were significantly higher than those in their controls. In the presence of Aa‐LPS, monocyte adhesion did not differ between treated and control 3D cultures but was significantly higher in treated 2D cultures than in the controls. In response to Pg‐LPS, cytokine‐prostaglandin secretion and monocyte adhesion did not differ between 3D and 2D cultures. These data indicate that HCAECs respond differently to these two types of LPS.

Modulating electrospun polycaprolactone scaffold morphology and composition to alter endothelial cell proliferation and angiogenic gene response

“…It has been shown in many cases that a 3D cell culture is favourable in relation to gene expression as it more accurately captures the morphological characteristics of the native ECM [51]. This has also been shown when using human coronary artery endothelial cells, where 3D culture showed a significant increase in the expression of key proteins compared to 2D culture [52].…”

Aggregatibacter actinomycetemcomitans and Atherosclerosis

“…However, the concentration of TNF-α, IL-7, IL-16, TNF-R1, IL-1ra and IL-1, are higher but not significant in 3D culture compared to control. The concentrations of all those soluble factors except for platelet-derived growth factor-BB are significantly higher in 3D-Aa-LPS than in 2D Aa-LPS [52].…”

“…Treatment with leukotoxin increased levels of both ICAM-1 and vascular cell adhesion protein 1 (106), which can result in rolling, adhesion and transmigration of monocytic cells into the vessel wall [50]. When endothelial cells are treated with Aa-derived LPS, the cytokines and chemokines (G-CSF, IL-8, RANTES, ICAM-1 and IL-6) secretion increase compared to untreated control [51,52]. However, the concentration of TNF-α, IL-7, IL-16, TNF-R1, IL-1ra and IL-1, are higher but not significant in 3D culture compared to control.…”

Aggregatibacter actinomycetemcomitans induces a proatherosclerotic response in human endothelial cells in a three‐dimensional collagen scaffold model

“…The use of type I collagen scaffolds prepared on the apical surface of Transwell inserts has previously been reported (Gualtero et al., 2019). However, in the present study, to avoid contraction of the scaffolds during lyophilization, the scaffolds were prepared with a higher concentration of collagen (10 mg/g w/w) than previously used.…”

“…Endotoxins stimulate the production of proinflammatory cytokines that positively regulate endothelial adhesion molecules and induce a prothrombotic environment (Li et al., 2000). Evidence from in vitro assays established a plausible mechanism of endothelial dysfunction in which periodontal pathogenic bacteria and their virulence factors induce a chronic proatherosclerotic inflammatory response in the endothelium, altering its normal function (Gualtero et al., 2019). Periodontal bacterial mechanisms to evade the host's immune response such as evasion of the complement system, inhibition of lymphocyte activity, and modulation of toll‐like receptor (TLR) can explain the survival of periodontopathogens outside the oral cavity (Reyes et al., 2013).…”