Clinopodium tomentosum (Kunth) Govaerts Leaf Extract Influences in vitro Cell Proliferation and Angiogenesis on Primary Cultures of Porcine Aortic Endothelial Cells

Abstract

Clinopodium tomentosum (Kunth) Govaerts is an endemic species in Ecuador, where it is used as an anti-inflammatory plant to treat respiratory and digestive affections. In this work, effects of a Clinopodium tomentosum ethanolic extract (CTEE), prepared from aerial parts of the plant, were investigated on vascular endothelium functions. In particularly, angiogenesis activity was evaluated, using primary cultures of porcine aortic endothelial cells (pAECs). Cells were cultured for 24 h in the presence of CTEE different concentrations (10, 25, 50, and 100 μg/ml); no viability alterations were found in the 10-50 μg/ml range, while a slight, but significant, proliferative effect was observed at the highest dose. In addition, treatment with CTEE was able to rescue LPS-induced injury in terms of cell viability. The CTEE ability to affect angiogenesis was evaluated by scratch test analysis and by an in vitro capillary-like network assay. Treatment with 25-50 μg/ml of extract caused a significant increase in pAEC's migration and tube formation capabilities compared to untreated cells, as results from the increased master junctions' number. On the other hand, CTEE at 100 μg/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25 μg/ml. The CTEE phytochemical composition was assessed through HPLC-DAD; rosmarinic acid among phenolic acids and hesperidin among flavonoids were found as major phenolic components. Total phenolic content and total flavonoid content assays showed that flavonoids are the most abundant class of polyphenols. The CTEE antioxidant activity was also showed by means of the DPPH and ORAC assays. Results indicate that CTEE possesses an angiogenic capacity in a dose-dependent manner; this represents an initial step in elucidating the mechanism of the therapeutic use of the plant.

Figure 1

Effect of CTEE on pAEC cell viability. Cell viability was measured by the MTT assay after treatment with different concentrations of CTEE. Data represent mean ± S.D. (n = 3). Different letters above the bars indicate significant differences (p < 0.05 ANOVA post hoc Tukey's test).

Figure 2

Effect of CTEE on LPS-induced pAEC damage. Cell viability was measured by the MTT assay after treatment with LPS (25 μg/ml) and different concentrations of CTEE. Data represent mean ± S.D. (n = 3). Different letters above the bars indicate significant differences (p < 0.05 ANOVA post hoc Tukey's test).

Figure 3

Dean-Jett-Fox Univariate cell cycle analysis by flow cytometry. Fluorescence of the PI-stained pAECs was measured using MACSQuant® Analyzer 10 and analyzed by Flowlogic software (Miltenyi Biotec, Bergisch Gladbach, Germany). 2 × 10⁵ cells were examined for each sample (n = 3), and the experiment was repeated twice. (a) Representative DNA content frequency histograms. (b) Cell cycle distribution for pAECs treated with various concentrations of CTEE (10, 25, 50, and 100 μg/ml) for 24 h in a grouped histograms graph. (^∗p < 0.0001; ^Δp < 0.001; ^□p < 0.01).

Figure 4

Effect of CTEE on pAEC migration capability. Cells were scratch wounded and then treated with CTEE at different concentrations (25, 50, and 100 μg/ml). Photographs were recorded at 18 h after scratching. (a) Representative microscopic phase-contrast pictures showing the size of the scratch wound in the 25 μg/ml CTEE-treated group compared with control. Scale bar, 100 μm. (b) Damaged area (percentage of the original scratch) as a function of different CTEE concentrations. Data represent mean ± S.D. (n = 3). Different letters above the bars indicate significant differences (p < 0.05 ANOVA post hoc Tukey's test).

Figure 5

CTEE effect on pAEC tube formation capability. pAECs were cultured on an extracellular matrix for 18 h with different concentrations of CTEE (25, 50, and 100 μg/ml). (a) Representative microscopic phase-contrast pictures showing capillary network in different treatment groups compared with control. Scale bar, 100 μm. (b) Number of master junctions in pAEC network. Data represent mean ± S.D. (n = 3). Different letters above the bars indicate significant differences (p < 0.05 ANOVA post hoc Tukey's test).

Figure 6

CTEE effect on VEGF and FLK-1 gene expressions. Relative expression (RE) was calculated as a fold of change with respect to the control cells; error bars represent the range of relative gene expression (n = 3). Different letters above the bars indicate significant differences (p < 0.05 ANOVA post hoc Tukey's test).