Effect of phloroglucinol derivatives isolated from <i>Ecklonia cava</i> on cell toxicity of EPCs.

<p>(A) Chemical structure of phloroglucinol: a class of natural products containing 1,3,5-trihydroxybenzene as the basic moiety. (B) Immunophenotyping of cell monolayers derived from HUCB EPCs by fluorescence microscope. A representative image is shown for HUCB EPCs. Immunophenotyping revealed that EPCs expressed endothelial cells lineage antigens including CD31, VEGFR-2 (KDR), VWF, eNOS, p-eNOS and p-Akt. (C) After treatment of phloroglucinol in EPCs, cell viability was examined using an MTT assay.</p

Effect of phloroglucinol on tubule-like structure formation of EPCs.

<p>(A) Representative tubular network structure of VEGF-stimulated outgrowths of ECs treated with or without phloroglucinol. Bevacizumab was used as a positive control. Bar: 500 µm. (B, C) Tube branches and total tube length were quantified using MacBiophotonics Images J software. Bar graph represents the number of intact loops in the capillary networks. Graph represents the length of tubes in the capillary networks (*P<0.05, **P<0.01).</p

Phloroglucinol attenuated VEGF-dependent angiogenesis a in matrigel plugs assay.

<p>(A) Experimental protocols used in the VEGF-dependent matrigel plug assay. (B) Representative matrigel plug of each group at 13 days after injection. C57BL/6 mice (n = 5 per group) were subcutaneously injected with growth factor-reduce matrigel alone (none) or a combination of VEGF (300 ng/ml) and phloroglucinol (0.94 mg/kg and 9.4 mg/kg). VEGF loaded plugs from mice exhibited red color indicating abundant red blood cells.</p

Effect of phloroglucinol on EPC mobilization in LLC tumor-bearing mice.

<p>(A) Schematic experimental schedule of <i>in vivo</i> EPC mobilization kinetics. After subcutaneous injection with 5×10⁶ LLC, the mice were orally administered DMSO vehicle (control) or 0.94 mg/kg phloroglucinol daily for 5 days after the initiation of therapy. Circulating MNCs were harvested at day 5 by Ficol-gradient centrifugation. (B) A subsequent gate was used to select the total CD45<i>^NEG</i> cell population. Corresponding flow cytometric analysis was used to detect CD34<i>^POS</i> cells in the gated CD45 <i>^NEG</i> cell population. The EPC population was represented as CD45<i>^NEG</i>/CD34<i>^POS</i> cells. (C) Statistical difference between phloroglucinol after oral administration of both phloroglucinol and vehicle in LLC-tumor bearing mice. Bar graph represents marked differences in EPC frequency at day 5 after daily injection of phloroglucinol or vehicle (*P<0.05).</p

Phloroglucinol decreased the number of CD31(+) vessels a in matrigel plugs assay.

<p>(A) Representative photomicrographs of CD31-stained matrigel sections obtained from mice treated with the vehicle. After 13 days, the mice were sacrificed, and the matrigel plugs were removed. The histological sections were fixed with 4% paraformaldehyde and embedded in paraffin. Infiltrating endothelial cells in plugs stained during an immunohistochemistry assay of anti-CD31 antibody. Bar: 500 µm. (B) Quantitative assessment of CD31-positive capillaries (*P<0.05, **P<0.01).</p

Effect of various concentrations of phloroglucinol on the migratory activity of EPCs in a wound healing assay.

<p>(A) <i>Ex vivo</i> cultured outgrowth ECs were subjected to a wound healing migration assay. Bar: 500 µm. EPCs were wounded and treated with 100 ng/ml of VEGF with or without 20 ìM or 100 ìM of phloroglucinol or a vehicle. (B) Bar graph represents the number of migrated cells. Fields were chosen randomly from various section levels to ensure objective sampling. In response to phloroglucinol, the VEGF-induced migratory activity of EPCs was significantly decreased.</p

Effect of phloroglucinol on tumor growth and tumor angiogenesis in LLC tumor-bearing mice.

<p>(A) Male C57BL/6 mice were injected subcutaneously with 5×10⁴ LLC cells. LLC tumor-bearing mice were treated with DMSO solvent control and phloroglucinol at 0.94 mg/kg daily for 24 days after initiation of therapy. (B) Tumor growth was measured with calipers every 3 or 4 days using the formula V = height×length×depth (cm³). All data are represented as the mean tumor volume ± SE for the 7 animals in each group.. (C) Representative photomicrographs of CD31 capillaries in tumor sections stained with rat anti-mouse CD31 (green fluorescence), a typical endothelial marker. Nuclei were counterstained with DAPI (blue). Sections were photographed at ×100 magnification using an fluorescent microscope. Bar: 500 µm, (D) Quantification of the density of CD31⁺ capillary neovessels. The number of CD31-stained capillaries was counted using the Image J program. Fields were chosen randomly from various section levels to ensure objective sampling (*P<0.05).</p

Genistein Promotes Endothelial Colony-Forming Cell (ECFC) Bioactivities and Cardiac Regeneration in Myocardial Infarction

<div><p>Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells <i>in vivo</i> because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model. <i>Results</i>: We found that genistein treatment enhanced ECFCs' migration and proliferation, which was accompanied by increases in the expression of ILK, α-parvin, F-actin, and phospholylation of ERK 1/2 signaling. Transplantation of genistein-stimulates ECFCs (GS-ECFCs) into myocardial ischemic sites <i>in vivo</i> induced cellular proliferation and secretion of angiogenic cytokines at the ischemic sites and thereby enhanced neovascularization and decreased myocardial fibrosis as well as improved cardiac function, as shown by echocardiography. Taken together, these data suggest that pretreatment of ECFCs with genistein prior to transplantation can improve the regenerative potential in ischemic tissues, providing a novel strategy in adult stem cell therapy for ischemic diseases.</p></div

ERK1/2-mediated genistein-induced ECFC proliferation and survival in the border zone of the left ventricular (LV) infarct at 3 days after myocardial infarction (MI).

<p>(A) ECFCs were pretreated with U0126 (ERK1/2 inhibitor, 10⁻⁶ M) for 30 min prior to 12 h of genistein treatment and then washed with PBS, fixed, stained, and analyzed by flow cytometry. Gates were manually configured to determine the percentage of cells in S phase based on DNA content (n = 5). *P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC). ECFCs were pretreated with U0126 for 30 min prior to a 12 h genistein (10⁻¹⁰ M) treatment, and the cells were transplanted into the ischemic region. (B) Proliferating cell nuclear antigen (PCNA) staining to detect ECFC proliferation. PCNA+ cell zone, <b>yellow-boxed area.</b> (Scale bar: 100 µm). (C) Quantification of PCNA-positive cells at 3 d after MI. (D) Co-immunofluorescent staining to detect proliferation (Ki67 [proliferation marker, red] and of hECFCs [human nuclear antigen (HNA)-positive cells, green] and DAPI [blue] for nuclear staining). <b>Arrows</b> indicate Ki67+ HNA+ DAPI+ cells. (E) Quantitative analysis of Ki67/HNA/DAPI triple-positive cells at 3 days after MI. (F) Coimmunofluorescent staining to detect apoptosis (caspase-3, apoptosis marker, green) and of hEPCs (HNA-positive cells, red) and DAPI (blue) by nuclear staining. <b>Arrows</b> indicate caspase-3+ HNA+ DAPI+ cells. (Scale bar: 20 µm). (G) Quantitative analysis of caspase-3/HNA/DAPI triple-positive cells at 3 days after MI. (n = 6) *P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC).</p

ILK, α-parvin and F-actin mediated genistein-induced ECFC migration.

<p>(A) ECFCs were treated with genistein for 0–24 h, and ILK, α-parvin and F-actin was detected by western blotting. (B) ECFCs were transfected with ILK, α-parvin, and TRIOBP (F-actin) small interfering RNA (siRNA) (ILK, α-parvin, and TRIOBP-specific siRNA; 200 pmol) for 24 h before genistein treatment and staining with Calcein AM. Fluorescence in the analytical zone was quantified with a plate reader. *P<0.05 vs. CTRL (indicates control genistein untreated ECFCs), **P<0.05 vs. genistein. (C) ECFCs were transfected with ILK siRNA (ILK-specific siRNA; 200 pmol) for 24 h before genistein (10⁻¹⁰ M) treatment, and the cells were injected into the tail veins of mice 30 min after left anterior descending (LAD) artery ligation. Staining of ECs with isolectin B4 (green) showed human nuclei antibody (HNA) (red)-positive cell incorporation into the border zone of left ventricular (LV) infarct at 3 days after myocardial infarction (MI) (Scale bar: 20 µm). <b>Inset</b> in higher magnification of the <b>yellow-boxed area</b>. <b>Arrows</b> indicate of isolectin B4+HNA+DAPI+cells. (D) The bar graph shows quantitative analysis of the number of HNA+cells associated with isolectin B4+vasculature (n = 5). HPF indicates high-power field. *P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC).</p