It is well recognized that vascular endothelial cells exert marked regulatory influence on subjacent non-endothelial cells such as myocytes, fibroblasts, pericytes and smooth muscle cells (SMCs) [9–13]. Given the regional differences in the functional properties of the endothelium, we had earlier investigated the role of EE in modulating cardiac fibroblast proliferation and collagen synthesis and found that EE has a stimulatory effect on cardiac fibroblasts . In the present study we explored whether pro – inflammatory agents such as TNF-α and LPS modulate this stimulatory effect. Interestingly, both TNF-α and LPS were found to attenuate the stimulatory effect of EE on cardiac fibroblasts.
The inflammatory effects of TNF-α and LPS on vascular endothelial cells are well – characterized. Endothelial cells obtained from different sites exhibit varied responses to cytokines and LPS . However, the effects of TNF-α and LPS on endocardial endothelial cells have not been previously reported.
In the present study, [3H]-Thymidine incorporation as well as rate of collagen synthesis were significantly lower in cardiac fibroblasts grown in conditioned medium from EECs treated with either TNF-α or LPS, when compared to the cells grown in EEC conditioned medium and not treated with either agents. Neither TNF-α nor LPS affected the viability of the cells. Concentrations of TNF-α up to 1000 ng/ml have been reported to cause reduction in collagen synthesis in cardiac fibroblasts without affecting the cell numbers . Our study demonstrated not only the direct inhibitory action of TNF-α on collagen synthesis in cardiac fibroblasts, but also its ability to attenuate the stimulatory effect of EECs on collagen production by cardiac fibroblasts. Yokoyama et al  proposed that TNF-α could act as an autocrine/paracrine mediator in myocardial remodeling. The cytokine increases both the expression and activity of matrix metalloproteinases (MMPs) which regulate matrix turnover . In studies investigating the direct effect of TNF-α on cardiac fibroblasts, it has been observed that the cytokine decreases total collagen synthesis [18, 19].
We also observed alterations in the release of endothelium-derived factors, such as NO, TGF-β and ET-1 into the conditioned medium when EECs were treated with TNF-a or LPS, which in turn could contribute to the altered response elicited in cardiac fibroblasts treated with the conditioned medium. EECs released significantly higher levels of nitrite in response to the pro-inflammatory agents. A notable action of TNF-α is its ability to induce nitric oxide synthase (NOS) activity in different cell types, including endocardial cells [6, 20]. Nitric oxide (NO) is an important modulator of TNF-α in the heart. In addition to being an immunomodulator and a potent inhibitor of platelet aggregation and cell migration, NO is also an anti-mitogen [21–23]. In a study using endothelial cells and SMCs from coronary arteries, inhibition of NO has been shown to cause an increase in the concentration of collagen types I and III. The data also supports an inhibitory role for NO on collagen synthesis . Extrapolating these findings to the present study, it is tempting to postulate that the attenuation of the proliferative response in cardiac fibroblasts is the result of increased release of NO from EECs on treatment with TNF-α or LPS.
Levels of endothelin (ET-1) in the conditioned medium from EECs treated with TNF-∝ or LPS were found to be lower, raising the possibility that a decrease in the levels of ET-1 may contribute to the diminished proliferative response in fibroblasts. Our earlier studies using endothelin inhibitors had shown that the stimulatory effect of EECs on cardiac fibroblasts is mediated by ET-1 .
Treatment of EECs with TNF-∝ also resulted in an elevation of TGF-β in the conditioned medium. Dose-dependent increase in TGF-β release has been observed in microglial cells treated with TNF-∝ . It is possible that TNF-∝ may regulate TGF-β expression as a feed back mechanism to limit extra cellular degradation in response to injury. TGF-β being inhibitory to cardiac fibroblast proliferation, the increased levels of this peptide in the conditioned medium from TNF-∝ treated EECs obviously accentuates the attenuation of proliferation brought about by increased NO and decreased ET-1.
LPS causes cardiac dysfunction by enhancing cardiac-derived inflammatory mediator expression, associated with the release of pro-inflammatory cytokines such as TNF-α and IL-1β and over production of NO [26, 27]. Human coronary endothelial cells stimulated with LPS express higher levels of TNF mRNA and release increased levels of the cytokine . Since most of the effects of LPS are through TNF, it can be assumed that in cardiac fibroblasts, the endotoxin elicits responses similar to that induced by TNF-α.
In this study, we have explored the independent effects of TNF-α and LPS on fibroblast function mediated through major mediators released by EECs. It is possible that other EE-derived mediators are also involved in the modulation of fibroblast growth and collagen synthesis. A limitation of the study is the usage of cells from two different species, but in previous studies on the interaction of endothelial cells with their neighboring cells, others have also employed cells derived from different species .