Functional and biochemical endothelial profiling in vivo in a murine model of endothelial dysfunction : comparison of effects of 1-methylnicotinamide and angiotensin-converting enzyme inhibitor

Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo. Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR(-/-)) in vivo, in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1–7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR(-/-) mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate(®)FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T(1)) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1–7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR(-/-) mice, Ach induced a vasoconstriction in BCA that amounted to –7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1–7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1–9) and Ang-(1–7) and a proportional decrease in Ang II and its active metabolites. Finally, MNA and perindopril treatment resulted in an increase in L-arginine/ADMA ratio by 107% (MNA) and 140% (perindopril), as compared to non-treated mice. Functional and biochemical endothelial profiling in ApoE/LDLR(-/-) mice in vivo revealed that 2-month treatment with MNA (100 mg/kg/day) displayed a similar profile of vasoprotective effect as 2-month treatment with perindopril (10 mg/kg/day): i.e., the improvement in endothelial function that was associated with the beneficial changes in ACE/Ang II-ACE2/Ang (1–7) balance and in L-arginine/ADMA ratio in plasma

Decreased serum essential and aromatic amino acids in patients with chronic pancreatitis

AIM: To investigate the influence of chronic pancreatitis (CP) on serum concentrations of amino acids

Hepatocyte Nuclear Factor 1α Proinflammatory Effect Linked to the Overexpression of Liver Nuclear Factor–κB in Experimental Model of Chronic Kidney Disease

Chronic kidney disease (CKD) is associated with low-grade inflammation that activates nuclear factor–κB (NF–κB), which upregulates the expression of numerous NF–κB responsive genes, including the genes encoding IL-6, ICAM-1, VCAM-1, and MCP-1. Herein, we found the coordinated overexpression of genes encoding RelA/p65 (a subunit of NF–κB) and HNF1α in the livers of chronic renal failure (CRF) rats—an experimental model of CKD. The coordinated overexpression of RelA/p65 and HNF1α was associated with a significant increase in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. A positive correlation between liver RelA/p65 mRNA levels and a serum concentration of creatinine and BUN suggest that RelA/p65 gene transcription is tightly related to the progression of renal failure. The knockdown of HNF1α in the HepG2 cell line by siRNA led to a decrease in Rel A/p65 mRNA levels. This was associated with a decrease in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. The simultaneous repression of HNF-1α and RelA/p65 by clofibrate is tightly associated with the downregulation of IL-6, ICAM-1, VCAM-1, and MCP-1 gene expression. In conclusion, our findings suggest that NF–κB could be a downstream component of the HNF1α-initiated signaling pathway in the livers of CRF rats

Differential involvement of IL-6 in the early and late phase of 1-methylnicotinamide (MNA) release in Concanavalin A-induced hepatitis

Exogenous 1-methylnicotinamide (MNA) displays anti-inflammatory activity. The aim of this work was to characterize the profile of release of endogenous MNA during the initiation and progression of murine hepatitis induced by Concanavalin A (ConA). In particular we aimed to clarify the role of interleukin-6 (IL-6) as well as the energy state of hepatocytes in MNA release in early and late phases of ConA-induced hepatitis in mice. Hepatitis was induced by ConA in IL-6+/+ and IL-6-/- mice, and various parameters of liver inflammation and injury, as well as the energy state of hepatocytes, were analysed in relation to MNA release. The decrease in ATP/ADP and NADH/NAD ratios, cytokine release (IL-6, IFN-γ), acute phase response (e.g. haptoglobin) and liver injury (alanine aminotransaminase, ALT) were all blunted in ConA-induced hepatitis in IL-6-/- mice as compared to IL-6+/+ mice. The release of MNA in response to Con A was also significantly blunted in IL-6-/- mice as compared to IL-6+/+ mice in the early stage of ConA-induced hepatitis. In turn, nicotinamide N-methyltransferase (NNMT) and aldehyde oxidase (AO) activities were blunted in the liver and MNA plasma concentration was elevated to similar degree in the late stage after Concanavalin A in IL-6+/+ and IL-6-/- mice. In conclusion, we demonstrated that in ConA-induced hepatitis, early, but not late MNA release was IL-6-dependent. Our results suggest that in the initiation and early hepatitis, MNA release is linked to the energy deficit/impaired redox status in hepatocytes, while in a later phase, MNA release is rather linked to the systemic inflammation

Enhanced muscle strength in dyslipidemic mice and its relation to increased capacity for fatty acid oxidation

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice

Innovative and Efficient Oral Delivery Method of APOA-1Milano Muteins Which Retain Anti-Atherosclerotic and Anti-Inflammatory Properties

Background. The management of modifiable risk factors exposure, in particular dyslipidemia, is the first line of intervention in preventing cardiovascular events. HDLs have been demonstrated to have anti-inflammatory and atheroprotective properties and, among HDLs, Apolipoprotein A-I (APOA-1), which promotes reverse cholesterol efflux, has been deeply investigated for the great therapeutic potential, particularly emphasized for the naturally occurring mutation APOA-1Milano (APOA-1M). Despite the high therapeutic potential of these molecules, their purification and delivery into the disordered organism is still limited by a very low efficiency in these processes. Aim. To develop an efficient system of production and delivery of APOA-1 muteins without need of purification. Methods and Results. Rice plants were genetically modified to express APOA-1M protein in their seeds. Protein extract from transgenic rice seeds (the ‘APOA-1M rice milk’) was tested for functionality in vitro on THP-1 macrophages exposed to oxLDL. Protein extract from wild type rice seeds (the ‘WT rice milk’) was used as control. The APOA-1M rice milk, but not the WT rice milk, significantly reduced expression of MCP-1 in oxLDL-loaded THP-1 macrophages in a dose-dependent manner and it promoted reverse cholesterol efflux in THP-1 macrophages. The lack of toxicity and the tolerability of the orally administered APOA-1M rice milk was evaluated in healthy mice. In an early-intermediate atherosclerotic mouse model (Apoe-/-mice fed with Western Diet for 8 weeks), 3 weeks of APOA-1M, but not the WT, rice milk treatment (15d, 5d/week, by oral gavage) significantly reduced area of lipid deposition and lipids concentration at aortic arch (en face analysis). Moreover, the APOA-1M, but not the WT, rice milk treatment reduced the hepatic CD68-positive cells and ameliorated the lipid management gene expression profile in liver of WD-fed Apoe-/-mice. Interestingly, all these findings were observed in mice still exposed to WD during the therapeutic treatment. Conclusion. The oral delivery of APOA-1M muteins, by means of genetically modified rice seeds extract, is athero-protective and anti-inflammatory even if the organism is exposed to high fat diet during the treatment, suggesting that this therapeutic approach could be effective in preventing and in counteracting atherogenic risk factors

APOA-1Milano muteins, orally delivered via genetically modified rice, show anti-atherogenic and anti-inflammatory properties in vitro and in Apoe-/- atherosclerotic mice

BACKGROUND: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes. METHODS AND RESULTS: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe-/- mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe-/- mice as compared to wild type rice milk-treated, WD-fed Apoe-/- mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe-/- mice as compared to WT rice milk treated mice. TRANSLATIONAL IMPACT: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the 'rice milk') to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies' limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients