Effect of Monocerin, a Fungal Secondary Metabolite, on Endothelial Cells

Abstract

This study reports the isolation and identification of the endophytic fungus Exserohilum rostratum through molecular and morphological analysis using optical and transmission electron microscopy (TEM), as well as the procurement of its secondary metabolite monocerin, an isocoumarin derivative. Considering the previously observed biological activities of monocerin, this study was performed on human umbilical vein endothelial cells (HUVECs) that are widely used as an in vitro model for several different purposes. Important parameters, such as cell viability, senescence-associated β-galactosidase, cellular proliferation by using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), apoptosis analysis with annexin, cellular morphology through scanning electron microscopy (SEM), and laser confocal analysis were evaluated after exposing the cells to monocerin. After 24 h of exposure to monocerin at 1.25 mM, there was more than 80% of cell viability and a low percentage of cells in the early and late apoptosis and necrosis. Monocerin increased cell proliferation and did not induce cell senescence. Morphological analysis showed cellular integrity. The study demonstrates aspects of the mechanism of action of monocerin on endothelial cell proliferation, suggesting the possibility of its pharmaceutical application, such as in regenerative medicine.

Keywords: cell integrity; cell viability; monocerin; proliferation; secondary metabolite; senescence.

Figure 1

Phylogenetic tree built by the neighbor-joining method using the Jukes and Cantor models for the ITS1-5,8S-ITS2 sequence of the endophytic strain FV3 used to produce the compound monocerin. The GenBank accession of the sequences used in this analysis is presented in Supplementary Material (Table S1).

Figure 2

Morphological features of the fungus E. rostratum obtained through optical microscopy ((A,B), bars of 50 and 20 µm), including an inset of the fungus colony picture (B), scanning electron microscopy ((C), 1720×, bar = 50 µm), and transmission electron microscopy ((D), 4646×, bar = 2 µm). Indication of the thickness wall, containing melanin, measured using TEM as 447.64 nm (Leo 906 E Zeiss microscopy serial # 9682, Kv maximum of 120 Kv).

Figure 3

Representation of endothelial cells’ (HUVECs) viability after treatment with monocerin from 0.02 to 1.25 mM for 24 h in six replicates in three independent assays (mean ± SE). The control group refers to untreated cells that receive only serum-free RPMI-1640 medium and were set as 100% of cell viability.

Figure 4

Proliferation index of endothelial cells (HUVECs) exposed to monocerin at 0.02 and 0.15 mM in triplicate for 24, 48, and 72 h. Data represent mean ± SE from two independent experiments compared to untreated cells (control). * p < 0.05. ** p < 0.01 and *** p < 0.001.

Figure 5

(A) Percentage of viable cells, in necrosis, and in initial and late apoptosis (mean ± SE). Histogram of double annexin/PI staining of untreated (control) endothelial cells (B) and following the treatment with monocerin at (C) 0.02, (D) 0.15, (E) 0.625, and (F) 1.25 mM. Quadrants: left lower = viable cells; right lower = apoptotic cells (annexin V+); upper right = late apoptotic cells (annexin V+ and PI+); upper left = necrotic cells (PI+). Cell distribution in viable, apoptotic (annexin V+), late apoptotic (annexin V+ and PI+), and necrotic cells (PI+). (*** p < 0.001).

Figure 6

(A) Percentage of senescence of endothelial cells treated with monocerin (Mean ± SE), as well as photomicrograph representatives of senescence processes for (B) untreated (control) cells and those treated with monocerin at (C) 0.02, (D) 0.15, (E) 0.625, and (F) 1.25 mM for 24 h. Cells in blue are senescent, and the brilliant colorless ones are non-senescent cells detected on the β-galactosidase assay. Bars = 100 µm.

Figure 7

Photomicrograph obtained through scanning electron microscopy of untreated (control) endothelial cells (A,B) (10 μm/9603× and 3 μm/30,024×), and under exposure to monocerin for 24 h at (C,D) 0.02 (10 μm/9578× and 4 μm/24,557×), (E,F) 0.15 (10 μm/7398× and 5 μm/16,992×), (G,H) 0.625 (10 μm/8409× and 3 μm/27,904×), and (I,J) 1.25 mM (5 μm/11,178× and 5 μm/11,028×). Control cells were adherent, willing to constitute a monolayer with the presence of extracellular matrix formation (red arrows), cytoplasmic prolongations, and adhesion to the surface (yellow arrows). Cell junction elements and microvillus are present (orange arrow). Granules of different sizes and forms are adhered and distributed around the cellular body surface for the control and the monocerin group (green arrow). HUVECs treated with monocerin showed retractions of their cytoplasms and inter-cell communication due to the induction of cell division (yellow circle). For monocerin at 0.625 mM, there is an apoptotic body in degeneration, showing holes with corrosive aspect (white arrow). For the higher concentration of 1.25 mM of monocerin, the cell adhesion was lost (blue arrow), and a loss of the microvillus can be observed. There is formation of cellular aggregates, apoptotic bodies (pink arrows), and bubbles around the membrane surface.

Figure 8

Photomicrographs obtained through laser confocal microscopy of untreated (control) endothelial cells (A) and treated with monocerin at (B) 0.02, (C) 0.15, (D) 0.625, and (E) 1.25 mM for 24 h and labeled with acridine orange, an indicator for acidic vacuoles in cells and lysosomes. For treatments with 0.02, 0.15, and 0.625 mM of monocerin, it is possible to observe cell division. For cells treated with monocerin, mainly at 0.625 mM, there is a nuclear modification with heterogeneous chromatin distribution and aggregation of DNA in the nucleus with lower fluorescence intensity. At 1.25 mM, there is disorganization of the nuclear material, with significant loss of cell density and prominences on the surface of the cell membrane and dead cells. Illustration for rhodamine 123 staining for (F) untreated cells, or treated with monocerin at (G) 0.02, (H) 0.15, (I) 0.625, and (J) 1.25 mM. Rhodamine staining shows homogeneous distribution of mitochondria in the cytoplasm. At 0.625 and 1.25 mM, there is nuclear pleomorphism and retraction of the cytoplasmatic expansions. Bars = 50 µm.