Protocatechuic aldehyde exhibits endothelial protection.

<p>(<b>A</b>) Re-endothelization of a Sprague Dawley rat carotid artery determined by Evans Blue staining (de-endothelized areas are stained with blue). Ratios of surface covered by endothelium to the total area in sham, vehicle, and injured (Inj) groups treated with different substances as follows: PCA, G1, G15, G15+PCA, G15+G1 (n = 4 each). (<b>B</b>) Cross sections of rat common carotid arteries stained with CD31 antibody 3 days after injury. Also, targets related to inflammatory markers were observed including VCAM-1, CD40 and GPER-1. Data are presented as mean±SEM; *** indicates P<0.001 compared to the sham group. ## and ### indicate P<0.005 and P<0.001, respectively, compared to the vehicle group. § indicates P<0.05 compared to the G15+ Inj. group.</p

The effects of PCA, G1, G15, G15+PCA, and G15+G1 in CCA balloon-injured Sprague Dawley rats.

<p>(<b>A, B</b>) Seven-week-old Sprague Dawley rats (200 g) were treated with each substance by intraperitoneal injection for 2 weeks and then common carotid arteries from the rats were balloon injured. Substances were injected for another 4 weeks and then sacrificed for (<b>A</b>) H & E staining of common carotid arteries. Graph shows the percentage of neointima areas of Sprague Dawley rats from each group (n = 7). Measurements were made using Scion Image software. (<b>B</b>) immunohistochemistry of rat aortas shows GPER-1 expression in the linings of the aorta from the same tissues used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113242#pone-0113242-g005" target="_blank">Figure 5A</a>. Data are presented as mean±SEM; *** indicates P<0.001 compared to the sham group. ### indicates P<0.001 compared to the vehicle group. §§§ indicates P<0.001 compared to the G15 + Inj. group.</p

PCA inhibits angiogenesis.

<p>(<b>A</b>) Rat aortas were obtained from Sprague Dawley rats weighing 100 grams. Rat aorta segments placed on Matrigel were stimulated with FBS (10%) for 3 days. Then, the segments were treated with each substance for 48 hrs in media supplemented with FBS (10%). Sprout lengths were measured by using Scion Image software. (<b>A, B</b>) Values represent the means ± SEMs of 3 experiments. *** indicates P<0.001 compared to the control group. ## and ### indicate P<0.005 and P<0.001, respectively, compared to the PDGF group. §§§ indicates P<0.001 compared to the G15 group. Production will need this reference to link the reader to the figure.</p

Protocatechuic aldehyde decreases ROS production.

<p>(<b>A</b>) HUVECs were pretreated for 24 hrs with various concentrations (10, 50, 100 µM) of PCA. (<b>B</b>) HUVECs were pretreated for 24 hrs with PCA (100 µM) and G1 (3.0 µM) and for 6 hrs with G15 (3.0 µM). Then, (<b>A, B</b>) HUVECs were treated with H₂O₂ (100 µM/ml) for 1 hr followed by measurement of ROS levels. (<b>A, B</b>) Both experiments are representative of 3 independent experiments. Data are presented as mean±SEM; * P<0.05, ** P<0.005, # indicates P<0.05 compared to the control group.</p

PCA induces expression of GPER-1.

<p>(<b>A</b>) The chemical structure of PCA and G1. (<b>B</b>) HUVECs were treated with G1 (3.0 µM) and PCA (10, 50, or 100 µM) for 1 hr to measure the levels of cAMP activity. (<b>C</b>) HUVECs were treated with PCA (100 µM) over 1, 3, 6, 24 hrs. (<b>D</b>) HUVECs were treated with PCA (10, 50, or 100 µM) for 6 hrs. Blots are representative of 3 independent experiments. Densitometric analyses are presented as the relative ratio of GPER-1 to β-actin. Data are presented as mean±SEM; * P<0.05. (<b>E</b>) Aortic segments were harvested from Sprague Dawley rats and cultured <i>ex vivo</i> in serum-free media with PCA (100 µM) over 1, 3, 6, 24 hrs. Segments were than fixed for immunohistochemistry of GPER-1 using 4% paraformaldehyde.</p

Human umbilical vein endothelial cells (HUVECs) treated with tumor necrosis factor alpha (TNFα) and G15 induces inflammation.

<p>(A, B) HUVECs were pretreated with PCA (100 µM) and G1 (3.0 µM) for 24 hrs. (A) treatment with TNFα (10 ng/ml) for 6 hrs. (B) treatment with TNFα (10 ng/ml) for 1 hr. (C) HUVECs were pre-treated with PCA (100 µM) for 24 hrs and then treated with G15 (3.0 µM) for 6 hrs. Blots are representative of 3 independent experiments. (A, B, C) Densitometric analyses are presented as the relative ratio of ICAM-1, VCAM-1, CD40, HIF-1α, GPER-1 or phospho-p38 to β-actin. (B) Densitometric analyses for phospho-NF-κB are presented as the relative ratio to NF-κB. Data are presented as mean±SEM; * P<0.05, ** P<0.005, *** P<0.001.</p

Mean values of MNC+ and LINE-1 hypomethylation stratified by smoking and drinking status.

<p>^a The frequency of MNC+ per 1000 BN cells</p><p>^b Drinking status was classified as either a current alcohol drinker (yes) or not (no).</p><p>Mean values of MNC+ and LINE-1 hypomethylation stratified by smoking and drinking status.</p

Causal model for age and LINE-1 hypomethylation prediction of MNC+.

<p>(Top) the path from age to MNC+. (Bottom) another path from age through LINE-1 hypomethylation to MNC+. Arrows connecting one variable to another represent unstandardized regression coefficients of the path. Solid lines are significant whereas dashed lines are not.</p

Characteristics of the participants.

<p>^a The frequency of MNC+ per 1000 BN cells</p><p>^b There was one missing value, and the total number of participants was 31.</p><p>^c Drinking status was classified as either a current alcohol drinker (yes) or not (no).</p><p>Characteristics of the participants.</p

Vasculoprotective Effects of 3-Hydroxybenzaldehyde against VSMCs Proliferation and ECs Inflammation

<div><p>3-hydroxybenzaldehyde (3-HBA) is a precursor compound for phenolic compounds like Protocatechuic aldehyde (PCA). From recent reports, PCA has shown vasculoprotective potency, but the effects of 3-HBA remain unclear. The aim of this study is to investigate the vasculoprotective effects of 3-HBA in endothelial cells, vascular smooth muscle cells and various animal models. We tested effects of 3-HBA in both vitro and vivo. 3-HBA showed that it prevents PDGF-induced vascular smooth muscle cells (VSMCs) migration and proliferation from MTS, BrdU assays and inhibition of AKT phosphorylation. It arrested S and G0/G1 phase of VSMC cell cycle in PI staining and it also showed inhibited expression levels of Rb1 and CD1. In human umbilical vein endothelial cells (HUVECs), 3-HBA inhibited inflammatory markers and signaling molecules (VCAM-1, ICAM-1, p-NF-κB and p-p38). For ex vivo, 3-HBA has shown dramatic effects in suppressing the sprouting from aortic ring of Spargue Dawley (SD) rats. In vivo data supported the vasculoprotective effects of 3-HBA as it inhibited angiogenesis from Matrigel Plug assay in C57BL6 mouse, prevented ADP-induced thrombus generation, increased blood circulation after formation of thrombus, and attenuated neointima formation induced by common carotid artery balloon injury of SD rats. 3-HBA, a novel therapeutic agent, has shown vasculoprotective potency in both in vitro and in vivo.</p></div