Diabetes and Its Cardiovascular Complications: Potential Role of the Acetyltransferase p300

Diabetes has been shown to accelerate vascular senescence, which is associated with chronic inflammation and oxidative stress, both implicated in the development of endothelial dysfunction. This condition represents the initial alteration linking diabetes to related cardiovascular (CV) complications. Recently, it has been hypothesised that the acetyltransferase, p300, may contribute to establishing an early vascular senescent phenotype, playing a relevant role in diabetes-associated inflammation and oxidative stress, which drive endothelial dysfunction. Specifically, p300 can modulate vascular inflammation through epigenetic mechanisms and transcription factors acetylation. Indeed, it regulates the inflammatory pathway by interacting with nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) or by inducing its acetylation, suggesting a crucial role of p300 as a bridge between NF-κB p65 and the transcriptional machinery. Additionally, p300-mediated epigenetic modifications could be upstream of the activation of inflammatory cytokines, and they may induce oxidative stress by affecting the production of reactive oxygen species (ROS). Because several in vitro and in vivo studies shed light on the potential use of acetyltransferase inhibitors, a better understanding of the mechanisms underlying the role of p300 in diabetic vascular dysfunction could help in finding new strategies for the clinical management of CV diseases related to diabetes

Calcimimetic R-568 vasodilatory effect on mesenteric vascular beds from normotensive (WKY) and spontaneously hypertensive (SHR) rats. Potential involvement of vascular smooth muscle cells (vSMCs).

The potential role of calcimimetics as vasculotropic agents has been suggested since the discovery that calcium sensing receptors (CaSRs) are expressed in cardiovascular tissues. However, whether this effect is CaSR-dependent or -independent is still unclear. In the present study the vascular activity of calcimimetic R-568 was investigated in mesenteric vascular beds (MVBs) isolated from Spontaneously Hypertensive rats (SHR) and the relative age-matched Wistar-Kyoto (WKY) control rats. Pre-constricted MBVs were perfused with increasing concentrations of R-568 (10 nM- 30 μM) resulting in a rapid dose-dependent vasodilatation. However, in MVBs from SHR this was preceded by a small but significant vasoconstriction at lowest nanomolar concentrations used (10-300 nM). Pre-treatment with pharmacological inhibitors of nitric oxide (NO) synthase (NOS, L-NAME), KCa channels (CTX), cyclo-oxygenase (INDO) and CaSR (Calhex) or the endothelium removal suggest that NO, CaSR and the endothelium itself contribute to the R-568 vasodilatory/vasoconstrictor effects observed respectively in WKY/SHR MVBs. Conversely, the vasodilatory effects resulted by highest R-568 concentration were independent of these factors. Then, the ability of lower R-568 doses (0.1-1 μM) to activate endothelial-NOS (eNOS) pathway in MVBs homogenates was evaluated. The Akt and eNOS phosphorylation levels resulted increased in WKY homogenates and Calhex significantly blocked this effect. Notably, this did not occur in the SHR. Similarly, vascular smooth muscle cells (vSMCs) stimulation with lower R-568 doses resulted in Akt activation and increased NO production in WKY but not in SHR cells. Interestingly, in these cells this was associated with the absence of the biologically active dimeric form of the CaSR thus potentially contributing to explain the impaired vasorelaxant effect observed in response to R-568 in MVB from SHR compared to WKY. Overall, these findings provide new insight on the mechanisms of action of the calcimimetic R-568 in modulating vascular tone both in physiological and pathological conditions such as hypertension

Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM

Evaluation of endothelial mediators involved in vascular effects of R-568 in MVB from WKY and SHR rats.

<p>(A) and (B) Representative tracings of vascular response to R-568 (10 nM—30 μM) in MVB from WKY (A) or SHR (B) pretreated with L-NAME, CTX and INDO are shown. Each symbol represents the start of a 4-min perfusion. (C) MVB isolated from 8-week old WKY (closed symbols) were stimulated with increasing concentrations of R-568 in the absence (circles) or in the presence of L-NAME (100 μM/20 min; squares), L-NAME+CTX (300 nM/20 min; triangles), L-NAME+CTX+INDO (10 μM/20 min; diamonds). Results are the mean ± SEM of 4 WKY independent experiments. (D) MVB isolated from 8-week old SHR (open symbols) were stimulated with increasing concentrations of R-568 in the absence (circles) or in the presence of L-NAME (100 μM/20 min; squares), L-NAME+CTX (300 nM/20 min; triangles), L-NAME+CTX+INDO (10 μ M/20 min; diamonds). Results are the mean ± SEM of 4 WKY and 4 SHR independent experiments. *p < 0.04 and ^#p < 0.05 <i>vs</i> respective CTRL curve.</p

Evaluation of signaling pathways activated by R-568 in MVB from WKY and SHR.

<p>MVB homogenates from experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202354#pone.0202354.g001" target="_blank">Fig 1</a> were subjected to immunoblotting for total and phosphorylated forms of Akt (phospho-Akt^Ser473), and eNOS (phospho-eNOS^Ser1177). Total β-actin levels were used as loading control. A representative set of immunoblots is shown for experiments that were repeated independently 3 times. Each bar represents the mean ± SEM of densitometric analysis for phosphorylated proteins normalized to their respective total forms. *p < 0.05 <i>vs</i> respective basal.</p

CaSR expression in WKY- and SHR-vSMVCs.

<p>(A) Representative morphometric aspect of confluent vSMC cultures from normotensive rats (WKY) and spontaneously hypertensive rats (SHR) (magnification x 10; scale bar: 300 μM). The cells were plated and morphologically examined three different times in the same experimental conditions. (B) Representative pattern of electrophoretic bands (170–100, 70–55 and 40–25 kDa) that results from immunoblotting analysis of CaSR expression in WKY- and SHR-vSMCs lysates and its negative (NC, HEK293 empty vector transfected control cell lysate) and positive (PC, HEK293 CaSR transiently transfected cell lysate) controls. (C) Representative images of α-SMA flow cytometry analysis in WKY-and SHR-vSMCs and secondary antibody alone (control).</p

Effect of R-568 on Akt activation and NO production in vSMCs from WKY and SHR rats.

<p>(A) vSMCs stimulated with 1μM of R568 (R) at different times (30 seconds, 1 minute, 2 minutes) in the presence or absence of Calhex 231 (C, 1 μM) were subjected to immunoblotting for total and phosphorylated forms of Akt (phospho-Akt^Ser473). Total β-actin levels were used as loading control. A representative set of immunoblots is shown for experiments that were repeated independently 3 times. Each bar represents the mean ± SD of densitometric analysis for phosphorylated proteins normalized to their respective total forms. *p < 0.02 <i>vs</i> respective basal (CTRL), **p < 0.05 R+C <i>vs</i> respective R. (B) NO production determined by conversion of L-[3H]-arginine into L-[3H]-citrulline in vSMCs stimulated with 1μM of R568 in the presence or absence of Calhex 231 (1 μM), data are expressed as pmol/NO/min/mgprottot (picomoles /Nitric Oxide/ minutes/ milligrams protein total). Each bar represents the mean ± SD of 3 independent experiments. *p < 0.02 R568 <i>vs</i> respective basal, **p < 0.01 R568+Calhex <i>vs</i> respective R568.</p

Effect of endothelium removal and CaSR inhibition on vascular response to R-568.

<p>(A) MVB isolated from 8-week old WKY (closed symbols) and SHR (open symbols) were stimulated with increasing concentrations of R-568 in the presence (circles) or in the absence (squares) of endothelium. Results shown are the mean ± SEM of 3 (WKY) and 3 (SHR) independent experiments. Data from each curve were normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202354#pone.0202354.g001" target="_blank">Fig 1</a>. (B) MVB isolated from 8-week old WKY (closed symbols) and SHR (open symbols) were stimulated with increasing concentrations of R-568 in the absence (circles) or after pretreatment with (diamonds) Calhex-231 (3 μM/20 min). Results are the mean ± SEM of 3 WKY and 3 SHR independent experiments. Data from each curve were normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202354#pone.0202354.g001" target="_blank">Fig 1</a>. *p < 0.05 <i>vs</i> respective CTRL curve.</p