Branched‐chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation

Abstract Branched‐chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids implicated in glucose metabolism and maintenance of correct brain function. Elevated BCAA levels can promote an inflammatory response in peripheral blood mononuclear cells. However, there are no studies analysing the direct effects of BCAA on endothelial cells (ECs) and its possible modulation of vascular function. In vitro and ex vivo studies were performed in human ECs and aorta from male C57BL/6J mice, respectively. In ECs, BCAA (6 mmol/L) increased eNOS expression, reactive oxygen species production by mitochondria and NADPH oxidases, peroxynitrite formation and nitrotyrosine expression. Moreover, BCAA induced pro‐inflammatory responses through the transcription factor NF‐κB that resulted in the release of intracellular adhesion molecule‐1 and E‐selectin conferring endothelial activation and adhesion capacity to inflammatory cells. Pharmacological inhibition of mTORC1 intracellular signalling pathway decreased BCAA‐induced pro‐oxidant and pro‐inflammatory effects in ECs. In isolated murine aorta, BCAA elicited vasoconstrictor responses, particularly in pre‐contracted vessels and after NO synthase blockade, and triggered endothelial dysfunction, effects that were inhibited by different antioxidants, further demonstrating the potential of BCAA to induce oxidative stress with functional impact. In summary, we demonstrate that elevated BCAA levels generate inflammation and oxidative stress in ECs, thereby facilitating inflammatory cells adhesion and endothelial dysfunction. This might contribute to the increased cardiovascular risk observed in patients with elevated BCAA blood levels.


| INTRODUCTION
Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids which are important components of proteins in human skeletal muscles. 1 BCAA also modulate glucose metabolism 2 and contribute to the maintenance of correct brain function. 3 Therefore, BCAA are used as supplements in states of malnutrition to prevent muscular cachexia in critical and oncological patients. 4 In addition, these amino acids are commonly used at high doses as nutritional supplements to potentially improve mental and physical performance and with the purpose of muscle building. 5,6 However, there are not solid performed studies about the potential toxicity of excessive or chronic BCAA supplementation.
Increased BCAA plasma concentrations have been found in several pathological conditions such as Maple syrup urine disease (MSUD) 7 and type 2 diabetes (T2DM) and obesity. [8][9][10] Importantly, highly elevated BCAA blood concentrations in MSUD patients are responsible for neurological damage, 11 and in T2DM and obesity, elevated BCAA blood concentrations are associated with insulin resistance 10,12 and were suggested as important predictors of future diabetes and positively associated with enhanced cardiovascular risk. 13,14 In fact, some authors proposed BCAA as biomarkers for vascular complications such as subclinical atherosclerosis or coronary artery disease. 15 However, the mechanisms involved are rather poorly understood.
Chronic low-grade inflammation 16,17 and oxidative stress 18,19 are major pathophysiological mechanisms involved in T2DM, obesity and atherosclerosis leading to insulin resistance, endothelial dysfunction and micro-and macro-vascular complications.
Increased reactive oxygen species (ROS) generation induces endothelial dysfunction by impairing the bioactivity of endothelial NO and promotes leucocyte adhesion, inflammation, thrombosis and smooth muscle cell proliferation-all processes that exacerbate atherosclerosis. NADPH oxidase and mitochondria are key sources of vascular oxidative stress involved in endothelial dysfunction in several cardiovascular pathologies. 20, 21 We recently demonstrated that in cultured human peripheral blood mononuclear cells (PBMC), high BCAA concentration promotes oxidative stress from NADPH oxidase and mitochondria, the release of pro-inflammatory cytokines mediated by the activation of the nuclear transcription factor-κB (NF-κB) and the migration of PBMC via the activation of the mammalian target of rapamycin (mTORC1) axis. 22 However, whether this also occurs in endothelial cells (ECs), and whether it might contribute to endothelial dysfunction, is unknown.
In this study, we hypothesized that BCAA-derived ROS and inflammation might be important contributors of abnormal vascular function. Therefore, we evaluated the direct effects of high BCAA levels on ECs and aorta and the possible mechanisms involved in such effects with particular emphasis on ROS generation and inflammation.

| Cell culture
Human vascular ECs were isolated from the macroscopically healthy part of intact saphenous veins harvested from patients undergoing high ligation of varicose veins as described. 23 The veins were rinsed with PBS 1×, opened longitudinally to expose the endothelium and put it in direct contact with enzyme solution containing 1 mg/mL of collagenase type I (Gibco) for 30 minutes at 37°C in a humidified atmosphere of CO 2 (5%). After the digestion step, the upper face of endothelium was scraped to detach the ECs. Then, cells were centrifuged and seeded on gelatin 0.5% coated 6-well dishes and maintained in DMEM-F12 medium supplemented with FBS (20%), endothelial cells growth factor (ECGF, 30 μg/mL) and heparin (0.1 mg/mL) all from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA) in a 37°C, 5% CO 2 humidified incubator. After 5-7 days in DMEM-F12, several cell colonies grew and were selected with human CD31 antibody bound to Dynabeads (Invitrogen, Life Technologies, Carlsbad, CA, USA). Cell cultures were used between passages 2 and 5. ECs were stimulated with BCAA (0.2-12 mmol/L or 6 mmol/L) for 1 hour in the presence or absence of different inhibitors (see Section 3) added 30 minutes before stimulation. Control cells were not exposed to stimuli or inhibitors.

| NADPH oxidase activity assay
The O 2 ·− production generated by NADPH oxidase activity was determined by a chemiluminescence assay, as described. 22  if they were exposed to a passive tension equivalent to that produced by a transmural pressure of 100 mm Hg. Contractility of the segments was tested by an initial exposure to a high K + solution (K + -KHS, 120 mmol/L). The presence of endothelium was determined by the ability of 10 μmol/L acetylcholine to relax arteries pre-contracted with phenylephrine at ∼50% K + -KHS contraction.
Thereafter, concentration-response curves to BCAA (0. In some experiments, mouse aortic segments were pre-incubated in the organ bath with N-nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L) in the absence or presence of gp91dstat, ML171, mito-TEMPO or celecoxib (1 μmol/L) before the BCAA concentration-response curves. These segments were not pre-contracted with phenylephrine. These drugs were added 30 minutes before L-NAME.
To demonstrate a possible direct effect of BCAA on vascular smooth muscle cells we performed experiments where the endothelium was mechanically removed. Concentration-response curves to BCAA in phenylephrine pre-contracted vessels or in arteries incubated or not with L-NAME were performed as described above.
In another set of experiments, aortic segments were exposed to BCAA (

| Immunohistochemistry
OCT-embedded aortic segments were stained using standard histology procedures. Immunostaining was carried out in 3-μm-thick tissue sections and fixed using phosphate-buffered 4% paraformaldehyde.
Endogenous peroxidase was blocked and aorta sections were incu-

| Fluorimetric peroxynitrite assay
Peroxynitrite levels were measured in supernatants from aortic seg-

| Statistical analysis
Results are expressed as mean ± standard error (SEM  PI3K/Akt pathway is as an upstream activator of mTORC1 in different cell types. 22,27,28 In ECs, BCAA promoted Akt phosphorylation ( Figure 1D). Interestingly, both the mTORC1 inhibitor rapamycin ZHENYUKH ET AL. (100 nmol/L) and the AMPK inducer AICAR decreased the BCAAinduced activation of Akt and mTORC1 ( Figure 1D,E) suggesting that in response to BCAA, AMPK is likely activated to counteract the Akt/mTOR pathways and that there is a reciprocal relationship between mTORC1 and Akt. Notably, BCAA also increased gene and protein eNOS expression ( Figure 1F).

| BCAA induce oxidative stress by activating the mTORC1 pathway
We next investigated whether BCAA induce oxidative stress in ECs and we focused on NADPH oxidase and mitochondria as major sources of ROS at vascular level. As shown in Figure 2A

| BCAA trigger NF-κB pro-inflammatory pathway in ECs
A positive relationship between oxidative stress generation and activation of the pro-inflammatory NF-κB pathway has been described in different clinical conditions. 29 One of the earliest events in NF-κB  Figure 3E).

| BCAA induce leucocyte adhesion to endothelium
The adhesion of monocytes to endothelial cells is considered one of the initial events in endothelial dysfunction in vascular pathologies. 31,32 In our recent study, high levels of BCAA induced PBMC migration 22 and herein we observed increased expression of adhesion molecules in ECs in response to BCAA. Thus, we next investigated the effect of BCAA on PBMC adhesion to ECs. As shown in To demonstrate the contribution of vascular smooth muscle cells to BCAA-induced contraction, we performed experiments where the endothelium was mechanically removed. As shown in Figure 5E, in endothelium-denuded arteries pre-contracted with phenylephrine BCAA still induced a contractile response. However, this contractile response was not observed neither in basal conditions nor in arteries incubated with L-NAME ( Figure 5F), suggesting that endothelium-F I G U R E 4 BCAA promote leucocytes adhesion to endothelium. Effect of BCAA (6 mmol/L, 1 h) on leucocytes adhesion to ECs pre-incubated with or without rapamycin (RAPA), AICAR, wortmannin (W), mito-TEMPO (MITO), gp91dstat (dstat), ML171 (ML) and BAY-11-7082 (BAY). Data are expressed as mean ± SEM. ***P < .001 vs Control (C). ≠ P < .05; ≠≠ P < .01 vs BCAA. n = 7 dependent and -independent mechanisms are responsible for BCAAinduced contraction. They also suggest complex mechanisms of smooth muscle contraction in response to BCAA as pre-contraction seems to be needed to achieve contraction.

ROS production
Oxidative stress is a well-known promoter of endothelial dysfunction. 33 We next evaluated the effect of long exposure (24 hour) to Excessive O 2 ·− can react with NO increasing peroxynitrite levels, a potent oxidant that leads to the nitration of tyrosine residues in tissue proteins. Accordingly, BCAA incubation increased aortic protein levels of nitrotyrosine and peroxynitrite formation that were prevented by ML171 and gp91dstat ( Figure 7A,B). Together, these results suggest that BCAA impairs NO availability, rather than NO signalling in vascular smooth muscle cells, by increasing ROS levels mainly from NADPH oxidase.

| DISCUSSION
The main findings of our study are that high BCAA concentrations can trigger oxidative stress and NF-κB activation and inflammation in ECs and in the vasculature thus likely contributing to the endothelial dysfunction and cardiovascular disease frequently observed in different pathological conditions associated to elevated BCAA levels.
There are few data about the relationship between elevated BCAA plasma levels and inflammation. In MSUD patients, high BCAA blood concentrations cause neurological damage associated with sustained inflammation including elevated serum levels of IL-1β, IL-6 and IFN-γ. 34 In normoglycemic women, insulin resistance was associated with increased serum BCAA concentrations, down-regulation of mitochondrial energy metabolism and increased expression of inflammation-related genes (CCL2-CCL5) in the adipose tissue. 35 Finally, our recent study demonstrated that elevated concentrations of BCAA induced inflammation and oxidative stress in PBMC. 22 Our results in ECs further support the role of BCAA as mediators of inflammation and ROS production and provide novel important information that connects this systemic and local inflammatory milieu with vascular damage. Thus, BCAA trigger ROS generation from NADPH oxidases and mitochondria and also promote a pro-inflammatory response characterized by increased NF-κB activation and subsequent up-regulation of inflammatory molecules such as iNOS and the adhesion molecules ICAM-1 and E-selectin, which facilitate inflammatory cell migration 22 and adhesion to ECs (present study).
Interestingly, mitochondrial ROS was responsible at least in part, for the NF-κB activation, but this transcription factor did not influence ROS generation in response to BCAA, suggesting that alternative pathways exist for BCAA-induced oxidative stress. Our data are in contrast with those published by D'Antona et al, 36 showing improved mitochondria biogenesis and decreased ROS production together with increased antioxidant defenses in middle-aged (16 months old) mice supplemented with a BCAA enriched mixture during 3 months. However, this approach is clearly different from the acute effects of BCAA evaluated in our study. It is also important to highlight that in this study, 36 44 In turn, this mechanism might prevent, at least in part, downstream mTORC1-induced ROS production, inflammation and cell adhesion. In agreement, an AMPK activator AICAR, is able to decrease these parameters in response to BCAA.
It is well accepted that unbalanced ROS production actively participate in alterations of vascular tone associated with various diseases, such as hypertension, diabetes or atherosclerosis. 33 Our data provide a proof-of-concept of the potential harmful effect of BCAA in the vascular endothelium. However, the physiological relevance of the concentrations of BCAA and times of exposure used in the present study remain to be established. Thus, the pro-oxidant and inflammatory effects of BCAA were observed at concentrations that could be reached in MSUD 7,27 or in daily BCAA supplementation in sportsmen, 5,6 but higher than those found in patients suffering from obesity or diabetes. 8,9 However, it is important to highlight that lower concentrations of BCAA (0.5-2 mmol/L) already increased vascular contraction which might add physiological relevance. On the other hand, chronic exposure to moderately elevated BCAA levels added to hyperglycaemia and pro-inflammatory conditions decrease the threshold of mTOR phosphorylation and increase ROS formation in PBMC 27,37 and we cannot discard that this mechanism might be also operating in endothelial cells.
Together, our findings provide mechanistic and functional evidence linking elevated levels of BCAA and endothelial dysfunction.
We demonstrate that elevated BCAA levels produce inflammation and oxidative stress in endothelial cells via mTORC1 pathway, therefore facilitating inflammatory cells adhesion. In turn, this pro-oxidant and inflammatory milieu facilitate vascular hypercontractility and F I G U R E 7 BCAA induce nitrotyrosine expression and increase peroxynitrite levels. Effect of BCAA (6 mmol/L, 24 h) pre-incubated 30 min with or without ML171 and gp91dstat (dstat) on A, protein nitrosylation levels in aortic sections and B, peroxinytrite levels measured in supernatants from aortic segments. Data are expressed as mean ± SEM. *P < .05 vs Control (C). ≠ P < .05 vs BCAA. n = 3-5 diminished endothelium-dependent relaxation (Figure 8). If chronic exposure to these BCAA is achieved, these early events might eventually converge in atherosclerosis and other cardiovascular complications.

ACKNOWLEDGEMENTS
Olha Zhenyukh was the recipient of a fellowship from Fundación Conchita Rábago.

CONF LICT OF I NTEREST
No conflicts of interest relevant to this article were reported.

ETHICS STATEMENT
The procedure was approved by the Research Ethics Committee of F I G U R E 8 Scheme demonstrating the possible relationship between branched-chain amino acids (BCAA), reactive oxygen species (ROS) and inflammation and its putative role in endothelial dysfunction and cardiovascular diseases. The influx of BCAA into endothelial cells is mediated by binding to specific nutrient transporters. In cytoplasm, BCAA activate PI3K-Akt/mTORC1 and AMPK signalling pathways. The BCAAdependent activation of these pathways seems to induce NADPH oxidase activation, mitochondrial oxidative stress and nuclear transcription factor-κB (NF-κB) leading to increased production of ROS and pro-inflammatory factors. BCAA-induced oxidative and pro-inflammatory status promote leucocytes migration and adhesion to the endothelium. In addition, ROS derived from mitochondria or through mechanisms involving NOX-1 and NOX-2 subunits of NADPH oxidase could reduce NO availability. These events, in turn, would induce endothelial dysfunction and vasoconstriction. All together, these vascular alterations might contribute to the development of cardiovascular diseases in clinical conditions associated with elevated levels of BCAA