Effects of (-)-epicatechin and derivatives on nitric oxide mediated induction of mitochondrial proteins

Article abstract of DOI:10.1016/j.bmcl.2013.05.079

Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and likely contributes to the development of cardiovascular diseases (CVD). The stimulation of mitochondrial function and/or biogenesis is seen as a means to improve the bioenergetic and metabolic status of cells and thus, reduce CVD. In this study we examined the capacity of the flavanol (-)-epicatechin and two novel derivatives to enhance mitochondrial function and protein levels in cultured bovine coronary artery endothelial cells. As nitric oxide production by endothelial cells is suspected in mediating mitochondria effects (including biogenesis), we also examined the dependence of responses on this molecule using an inhibitor of nitric oxide synthase. Results indicate that the flavanol (-)-epicatechin and derivatives are capable of stimulating mitochondrial function as assessed by citrate synthase activity as well as induction of structural (porin, mitofilin) and oxidative phosporylation protein levels (complex I and II). Effects were blocked by the use of the chemical inhibitor of the synthase thus, evidencing a role for nitric oxide in mediating these effects. The results observed indicate that the three agents are effective in enhancing mitochondria function and protein content. The effects noted for (-)-epicatechin may serve to explain the healthy effects on cardiometabolic risk ascribed to the consumption of cocoa products.

Full text of DOI:10.1016/j.bmcl.2013.05.079

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4441–4446
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effects of (ꢀ)-epicatechin and derivatives on nitric oxide mediated
induction of mitochondrial proteins
Aldo Moreno-Ulloa a,b, Armando Cid a, Ivan Rubio-Gayosso b, Guillermo Ceballos a,b, Francisco Villarreal a,
Israel Ramirez-Sanchez a,b,
⇑
a University of California, San Diego, Department of Medicine, La Jolla, CA 92093-0613, USA
b Escuela Superior de Medicina, Instituto Politecnico Nacional, Seccion de Estudios de Posgrado e Investigacion, Escuela Superior de Medicina, IPN, Plan de San Luis y, Diaz Miron S/N,
Casco de Santo Tomas, Del. M. Hidalgo Mexico, DF 11340, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and likely
contributes to the development of cardiovascular diseases (CVD). The stimulation of mitochondrial func-
tion and/or biogenesis is seen as a means to improve the bioenergetic and metabolic status of cells and
thus, reduce CVD. In this study we examined the capacity of the flavanol (ꢀ)-epicatechin and two novel
derivatives to enhance mitochondrial function and protein levels in cultured bovine coronary artery
endothelial cells. As nitric oxide production by endothelial cells is suspected in mediating mitochondria
effects (including biogenesis), we also examined the dependence of responses on this molecule using an
inhibitor of nitric oxide synthase. Results indicate that the flavanol (ꢀ)-epicatechin and derivatives are
capable of stimulating mitochondrial function as assessed by citrate synthase activity as well as induction
of structural (porin, mitofilin) and oxidative phosporylation protein levels (complex I and II). Effects were
blocked by the use of the chemical inhibitor of the synthase thus, evidencing a role for nitric oxide in
mediating these effects. The results observed indicate that the three agents are effective in enhancing
mitochondria function and protein content. The effects noted for (ꢀ)-epicatechin may serve to explain
the healthy effects on cardiometabolic risk ascribed to the consumption of cocoa products.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 20 March 2013
Revised 6 May 2013
Accepted 13 May 2013
Available online 1 June 2013
Keywords:
Epicatechin
Mitochondria
Nitric oxide
Endothelial cells
Cardiovascular diseases (CVD) are a leading cause of mortality
and morbidity worldwide. The health of the endothelium is central
to the underlying pathophysiology of manyCVD.1 Endothelial health
is partly dependent on the physiological production of nitric oxide
(NO) mainly by the endothelial nitric oxide synthase (eNOS).2 Dis-
ruption in the bioavailability of NO facilitates a vasoconstricting
and procoagulating state characteristic of many CVD. Impaired
mitochondrial function represents an early manifestation of endo-
thelial dysfunction and likely contributes to the development of
CVD.3,4 Physiological increases in NO have been linked with mito-
chondrial biogenesis and thus, mitochondrial function.5–8 Therefore,
strategies that increase physiological NO production may enhance
mitochondrial biogenesis in endothelial and other cell types and
are promising for the preservation of cardiovascular health.
In vitro and in vivo studies suggest that the flavan-3-ol (ꢀ)-epi-
catechin (EPI) can exert CVD protection,9–11 potentially via
improving mitochondrial structure/function.12–14 These effects
have been partly ascribed to the capacity of EPI to activate eNOS
and thereby increase NO production.15,16 As previously reported
by us, a possible means by which EPI (and possibly derivatives)
triggers these responses can involve a putative receptor entity, as
there is indirect evidence for its presence.11,15 Under physiological
conditions, native EPI is prone to oxidation secondary to the reac-
tivity of the phenolic hydroxyl groups (OH).17 The pharmacological
effects of native EPI are also limited by its susceptibility to phase II
metabolism (sulfation and glucuronidation of the OH groups),
which can lead to a short half-life.18 We propose that the develop-
ment of more stable derivatives of EPI is a useful strategy to retain
biological activity and enhance potency.
Two strategies may be useful in protecting EPI from oxidation.
First, EPI in vitro and in vivo is mono-methylated by the endothe-
lium in the –OH of the catechol ring (ring B, see Fig. 1), leading to a
mixture of 3’- and 40-O-methyl epicatechin,19,20 which stabilizes
the molecule against oxidation. Although, 30-O-methyl epicatechin
(30-O-MeEPI) and EPI have different H-donating potentials, they
appear to posses the same biological capacities. For example, both
molecules inhibit cell death induced by H2O2, implying a mecha-
nism of action independent of their antioxidant properties as Spen-
cer et al proposed.21 Second, the acetylation of flavanols such as
(ꢀ)-epigallocatechin gallate confers protection to all OH groups
and improves the chemical stability of the compound under phys-
iological conditions,22 which translates into enhanced biological
actions.23
⇑
Corresponding author. Tel.: +52 55 5729 6000x62820.
E-mail address: israel.ramirez14@hotmail.com (I. Ramirez-Sanchez).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.079

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A. Moreno-Ulloa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4441–4446
Figure 1. Chemical structures of (ꢀ)-epicatechin and derivatives. Bold letters and numbers correspond to the ring position and carbon atom numbering, respectively.
The goal of the current study was to rationally develop com-
pounds derived from EPI with improved effects on NO production
and thence on mitochondrial enzyme activity and protein content.
We hypothesized that EPI acetylation or alkylation will confer sta-
bility to the molecule and thereby increase its efficacy on endothe-
lial NO production and NO-mediated effects on mitochondrial
endpoints. To test our hypothesis, we modified EPI via alkylation
or acetylation of OH groups. For our first approach, we used an
ethyl group as the alkylating agent, which mimics (but with an
additional –CH2) the biotransformation of EPI into 30-O-MeEPI,
which should yield similar or enhanced biological activity as EPI,
likely with improved stability. For our second approach, we acety-
lated all OH groups of EPI. The three compounds used were tested
for biological activity using a cell culture model of bovine coronary
artery endothelial cells (BCAEC).
For the acetylation modification, all OH groups of EPI were acet-
ylated, leading to 3,30,40,5,7-O-penta-acetyl (ꢀ)-epicatechin (PEPI).
The chemical structures of PEPI and Ethyl EPI are illustrated in
Figure 1. For the alkylation modification, EPI was ethylated at
the position 30 (ring B), leading to 30-O-ethyl (ꢀ)-epicatechin (Ethyl
EPI) (for synthesis procedures and spectra characterization see
Supplementary data). In all experiments, EPI was used as a positive
control. Administration of EPI to cultured endothelial cells stimu-
lates NO synthesis secondary to the activation of eNOS; the effect
Figure 2. Dose response effects of EPI, Ethyl EPI and PEPI on nitric oxide (NO)
levels. BCAEC (in triplicate) were treated at different doses with each compound
and media collected 10 min after treatment. Nitric oxide (nitrite/nitrate, nmol/mg
protein) levels were assessed by kit (Cayman Inc.). Values represent means of
triplicate assays and sigmoidal curves were generated by Prizm 5 (Graphpad Inc.).
peaks at 10 min and 1
l
M EPI.15 As a prelude to determine the ef-
by ꢁ30% (Fig. 3A). This increase in CS suggests an augmented endo-
thelial mitochondrial activity and/or density elicited by the epicat-
echin compounds. EC50 concentrations of PEPI, Ethyl EPI and EPI
achieved similar increases in activity.
fects of NO on mitochondria-related endpoints, we generated con-
centration-response curves of NO production for EPI, PEPI, and
Ethyl EPI (Fig. 2). The rank order of potency (EC50) derived from
the curves was PEPI (0.006 nmol/L) > Ethyl EPI (1 nmol/L) > EPI
(2 nmol/L) with respect to their capacities to increase NO produc-
tion in BCAEC.
The regulatory role of NO on mitochondrial structure/function
has been previously studied.24,25 The sustained exposure of cells
to NO can trigger mitochondrial biogenesis.5 However, mitochon-
drial biogenesis also appears to be only partly dependent on NO
signaling.5,8,26–29 We used the EC50 concentrations to evaluate ef-
fects of these compounds on mitochondrial endpoints over a 48 h
treatment. We first examined citrate synthase activity (CS). Citrate
synthase is the initial enzyme of the tricarboxylic acid (TCA) cy-
cle.30 CS has been routinely used to assess the oxidative and respi-
ratory capacity as well as mitochondrial volume and density in
cells and tissues.31–33 All compounds significantly increased CS
We then examined for changes in constitutive members of
mitochondrial structure, specifically, porin, the most abundant
outer mitochondrial membrane protein that often is used as a mar-
ker for cellular mitochondrial mass.34 PEPI and Ethyl EPI but not
EPI significantly increased porin levels over control (Fig. 3B). We
also examined changes in mitofilin, a protein from the inner mito-
chondrial membrane that promotes/induces cristae and cristae
junction formation.35 EPI and PEPI significantly increase mitofilin
protein levels (Fig. 3C), suggesting augmented inner mitochondrial
mass. To assess changes in the oxidative phosphorylation system,
we assessed levels of mitochondrial complex I (CI), the major entry
point for electrons from NADH produced by the TCA cycle.36 EPI,
and PEPI, stimulated CI by ꢁ20% and 60% over control, respec-
tively, while Ethyl EPI produced no significant effect (Fig. 3D).

A. Moreno-Ulloa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4441–4446
4443
C Mitofilin
Porin
GAPDH
39 KDa
37 KDa
78 KDa
A
B
GAPDH
20 KDa
D
CI
GAPDH
E
CII
GAPDH
30 KDa
Figure 3. Effects of EPI, Ethyl EPI and PEPI on BCAEC citrate synthase activity and mitochondrial protein levels. (A) CS was evaluated in BCAEC treated with either vehicle
(control), 2 nmol/L EPI, 1 nmol/L Ethyl EPI, or 0.006 nmol/L PEPI for 48 h. BCAEC; bovine coronary artery endothelial cells, CS; citrate synthase activity, EPI; (ꢀ)-epicatechin,
Ethyl EPI; 30-O-ethyl (ꢀ)-epicatechin, PEPI; 3,30,40,5,7-O-penta-acetyl (ꢀ)-epicatechin. Western blots were probed with specific antibodies against (B), Porin; (C), Mitofilin;
(D), complex I, and (E), complex II. Values were normalized by arbitrarily setting the densitometry of control levels to 100. Values are mean SEM; n = 3 per treatment. ⁄p
W
<0.05 vs. control, p <0.01 vs. control.
Mitofilin
78 KDa
A
B
GAPDH
37 KDa
CI
GAPDH
20 KDa
30 KDa
C
D
CII
GAPDH
Figure 4. Effects of L-NAME on EPI-induced increases in BCAEC citrate synthase activity and mitochondrial protein levels. CS (A) and mitofilin (B), complex I (C), complex II
(D) protein densities in BCAEC following exposure for 48 h to either vehicle (control), 2 nmol/L EPI, or 300 lmol/L L-NAME + 2 nmol/L EPI. BCAEC; bovine coronary artery
endothelial cells, CS; citrate synthase activity, EPI; (ꢀ)-epicatechin, L-NAME; L-NG-Nitroarginine methyl ester. Values were normalized by arbitrarily setting the densitometry
of control levels to 100. Values are mean SEM; n = 3 per treatment. ⁄p <0.05 vs. control, p <0.05 EPI vs. EPI + L-NAME.
W

4444
A. Moreno-Ulloa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4441–4446
A
B
39 KDa
37 KDa
Porin
GAPDH
Figure 5. Effects of L-NAME on Ethyl EPI-induced changes on citrate synthase activity and mitochondrial protein levels. CS (A) and porin (B) protein densities in BCAEC
following exposure for 48 h to either vehicle (control), 1 nmol/L Ethyl EPI, or 300 lmol/L L-NAME + 1 nmol/L Ethyl EPI. BCAEC; bovine coronary artery endothelial cells, CS;
citrate synthase activity, Ethyl EPI; 30-O-ethyl epicatechin, L-NAME; L-NG-Nitroarginine methyl ester. Values were normalized by arbitrarily setting the densitometry of
control levels to 100. Values are mean SEM; n = 3 per treatment. ⁄p <0.05 vs. control, p <0.05 Ethyl-EPI vs. Ethyl-EPI + L-NAME.
W
78 KDa
Porin
GAPDH
Mitofilin
39 KDa
C
GAPDH
37 KDa
A
B
D
E
GAPDH
30 KDa
CII
20 KDa
CI
GAPDH
Figure 6. Effects of L-NAME on PEPI-induced changes on citrate synthase activity and mitochondrial protein levels. CS (A) and porin (B), mitofilin (C), complex I (D), complex
II (E) protein densities in BCAEC following exposure for 48 h to either vehicle (control), 0.006 nmol/L PEPI, or 300 lmol/L L-NAME + 0.006 nmol/L PEPI. BCAEC; bovine
coronary artery endothelial cells, CS; citrate synthase activity, PEPI; 3,30,40,5,7-O-penta-acetyl (ꢀ)-epicatechin, L-NAME; L-NG-Nitroarginine methyl ester. Values were
normalized by arbitrarily setting the densitometry of control levels to 100. Values are mean SEM; n = 3 per treatment. ⁄p <0.05 vs. control, p <0.05 EPI vs. EPI + L-NAME.
W
Complex II (CII) was also evaluated as a direct link between the
TCA cycle and the respiratory chain,37 EPI and PEPI increased CII
protein levels by ꢁ25% increase over control (Fig. 3E). Thus, PEPI
treatment increased all the mitochondrial endpoints measured;
EPI and Ethyl EPI appear to exert a semi-selective effect on specific
mitochondrial proteins. Perhaps, a higher concentration is needed

A. Moreno-Ulloa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4441–4446
4445
TFAM
TFAM
GAPDH
~ 27 KDa
A
B
37 KDa
GAPDH
C
TFAM
GAPDH
Figure 7. Effects of EPI, Ethyl EPI and PEPI and L-NAME on BCAEC TFAM levels. BCAEC were treated with either vehicle (control), 2 nmol/L EPI, 1 nmol/L Ethyl EPI or
0.006 nmol/L PEPI for 48 h L-NAME (300 lM). Western blots were probed with specific antibodies against TFAM. (A) Effects of EPI, Ethyl-EPI and PEPI, (B) Effects of EPI L-
NAME and (C) Effects of PEPI L-NAME. EPI; (ꢀ)-epicatechin, Ethyl EPI; 30-O-ethyl (ꢀ)-epicatechin, PEPI; 3,30,40,5,7-O-penta-acetyl (ꢀ)-epicatechin. Values were normalized
by arbitrarily setting the densitometry of control levels to 100. Values are mean SEM; n = 3 per treatment. ⁄p <0.01 p <0.01 vs. control.
W
used the nitric oxide synthase (NOS) inhibitor L-NG-Nitroarginine
methyl ester (L-NAME). Treatment of BCAEC for 48 h with L-NAME
20 KDa
CI
37 KDa
by itself induced no significant change but blocked the effects of
EPI, PEPI, and Ethyl EPI on CS and all assessed mitochondrial pro-
tein levels (porin, mitofilin, CI and CII) (Figs. 4–6). To evidence the
nature of the effects on a recognized mitochondria biogenesis
related endpoint and the role played by NO, we also assessed the
effects of treatments on a key regulator of mitochondria gene
transcription, TFAM.38 Results indicate that EPI and PEPI induce
significant increases in TFAM and effects are blocked by L-NAME
(Fig. 7). The complete blockade of compound effects on mitochon-
drial endpoints by L-NAME strongly evidence the critical role that
NO production may play in regulating mitochondrial biology in
endothelial cells and thus, CVD. To further explore the relative role
played by NO on mitochondrial protein (CI) levels induced by the
compounds, we use a dose of each that yielded a similar induction
in NO levels. As shown in Figure 8, EPI and PEPI led to significant
increases in CI levels. A possible means by which the compounds
yield variable effects may be explained by different diffusion prop-
erties and/or selective metabolism.
We provide evidence that ethyl O-alkylation of EPI’s B ring pro-
duces a more potent stimulant of NO production that can trigger
significant increases in mitochondrial enzyme activity and protein
levels. Surprisingly, the acetylation of all the EPI OH groups nota-
bly increased the potency on NO production vs. EPI and Ethyl EPI
translating into significant increases in some but not all mitochon-
drial endpoints that we assessed. Thus, PEPI may represent a par-
ticular effective way of targeting NO production and mitochondrial
activity at very low concentrations; however, more work is neces-
sary to examine its in vivo metabolism since it could be rapidly
GAPDH
Figure 8. Effects of EPI, Ethyl EPI and PEPI on BCAEC mitochondrial complex I
protein levels. BCAEC were treated with either vehicle (control), 100 nmol/L EPI,
0.01 nmol/L Ethyl EPI or 0.01 nmol/L PEPI for 48 h. Western blots were probed with
specific antibodies against complex I (CI). EPI; (ꢀ)-epicatechin, Ethyl EPI; 30-O-
ethyl (ꢀ)-epicatechin, PEPI; 3,30,40,5,7-O-penta-acetyl (ꢀ)-epicatechin. Values were
normalized by arbitrarily setting the densitometry of control levels to 100. Values
are mean SEM; n = 3 per treatment. ⁄p <0.01, p <0.01 vs. control.
W
for EPI and Ethyl EPI to increase all the mitochondrial endpoints,
as we only used the EC50 for NO production.
In order to determine the role of EPI and derivatives stimulated
NO in mediating compound effects on mitochondrial endpoints, we

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11. Ramirez-Sanchez, I.; Nogueira, L.; Moreno, A.; Murphy, A.; Taub, P.; Perkins, G.;
Ceballos, G. M.; Hogan, M.; Malek, M.; Villarreal, F. J. Cardiovasc. Pharmacol.
2012, 60, 429.
12. Steffen, Y.; Schewe, T.; Sies, H. Biochem. Biophys. Res. Commun. 2005, 331, 1277.
13. Taub, P. R.; Ramirez-Sanchez, I.; Ciaraldi, T. P.; Perkins, G.; Murphy, A. N.;
Naviaux, R.; Hogan, M.; Maisel, A. S.; Henry, R. R.; Ceballos, G.; Villarreal, F. Clin.
Transl. Sci. 2012, 5, 43.
14. Nogueira, L.; Ramirez-Sanchez, I.; Perkins, G. A.; Murphy, A.; Taub, P. R.;
Ceballos, G.; Villarreal, F. J.; Hogan, M. C.; Malek, M. H. J. Physiol. 2011, 589,
4615.
hydrolyzed by intracellular and membrane-bound esterases, which
may in turn release EPI. In other words, PEPI may represent a
pro-drug form of EPI, which could be highly favorable for drug
development. The testing of these compounds for mitochondrial
targeting is warranted for diseases whose impaired cellular
bioenergetics are an important part of the pathophysiology of
endothelial dysfunction and/or CVD.
15. Ramirez-Sanchez, I.; Maya, L.; Ceballos, G.; Villarreal, F. Hypertension 2010, 55,
1398.
Acknowledgements
16. Brossette, T.; Hundsdorfer, C.; Kroncke, K. D.; Sies, H.; Stahl, W. Eur. J. Nutr.
2011, 50, 595.
17. Zhu, Q. Y.; Holt, R. R.; Lazarus, S. A.; Ensunsa, J. L.; Hammerstone, J. F.; Schmitz,
H. H.; Keen, C. L. J. Agric. Food Chem. 2002, 50, 1700.
18. Richelle, M.; Tavazzi, I.; Enslen, M.; Offord, E. A. Eur. J. Clin. Nutr. 1999, 53, 22.
19. Steffen, Y.; Gruber, C.; Schewe, T.; Sies, H. Arch. Biochem. Biophys. 2008, 469,
209.
20. Ottaviani, J. I.; Momma, T. Y.; Kuhnle, G. K.; Keen, C. L.; Schroeter, H. Free Radic.
Biol. Med. 2012, 52, 1403.
We would like to acknowledge the editorial help provided by
Dr. Laurence Brunton. Study was supported by NIH R24
DK092154, R01 HL43617 and P60 MD00220 provided to Dr. F. Vil-
larreal, CONACyT research grant #129889 and an unrestricted gift
to Dr. Ceballos by Cardero Therapeutics Inc.
21. Spencer, J. P.; Schroeter, H.; Kuhnle, G.; Srai, S. K.; Tyrrell, R. M.; Hahn, U.; Rice-
Evans, C. Biochem. J. 2001, 354, 493.
Supplementary data
22. Huo, C.; Wan, S. B.; Lam, W. H.; Li, L.; Wang, Z.; Landis-Piwowar, K. R.; Chen, D.;
Dou, Q. P.; Chan, T. H. Inflammopharmacology 2008, 16, 248.
23. Landis-Piwowar, K. R.; Huo, C.; Chen, D.; Milacic, V.; Shi, G.; Chan, T. H.; Dou, Q.
P. Cancer Res. 2007, 67, 4303.
24. Erusalimsky, J. D.; Moncada, S. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 2524.
25. Carreras, M. C.; Franco, M. C.; Peralta, J. G.; Poderoso, J. J. Mol. Aspects Med.
2004, 25, 125.
26. Chowanadisai, W.; Bauerly, K. A.; Tchaparian, E.; Wong, A.; Cortopassi, G. A.;
Rucker, R. B. J. Biol. Chem. 2010, 285, 142.
27. Csiszar, A.; Labinskyy, N.; Pinto, J. T.; Ballabh, P.; Zhang, H.; Losonczy, G.;
Pearson, K.; de Cabo, R.; Pacher, P.; Zhang, C.; Ungvari, Z. Am. J. Physiol. Heart
Circ. Physiol. 2009, 297, H13.
28. Funk, J. A.; Odejinmi, S.; Schnellmann, R. G. J. Pharmacol. Exp. Ther. 2010, 333,
593.
29. Rasbach, K. A.; Schnellmann, R. G. J. Pharmacol. Exp. Ther. 2008, 325, 536.
30. Raimundo, N.; Baysal, B. E.; Shadel, G. S. Trends Mol. Med. 2011, 17, 641.
31. Hoppeler, H. Int. J. Sports Med. 1986, 7, 187.
32. Spina, R. J.; Chi, M. M.; Hopkins, M. G.; Nemeth, P. M.; Lowry, O. H.; Holloszy, J.
O. J. Appl. Physiol. 1996, 80, 2250.
33. Holloszy, J. O.; Oscai, L. B.; Don, I. J.; Mole, P. A. Biochem. Biophys. Res. Commun.
1970, 40, 1368.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.05.
079.
References and notes
1. Versari, D.; Daghini, E.; Virdis, A.; Ghiadoni, L.; Taddei, S. Diabetes Care 2009,
32(Suppl. 2), S314.
2. Napoli, C.; Ignarro, L. J. Arch. Pharm. Res. 2009, 32, 1103.
3. Ballinger, S. W. Free Radic. Biol. Med. 2005, 38, 1278.
4. Shenouda, S. M.; Widlansky, M. E.; Chen, K.; Xu, G.; Holbrook, M.; Tabit, C. E.;
Hamburg, N. M.; Frame, A. A.; Caiano, T. L.; Kluge, M. A.; Duess, M. A.; Levit, A.;
Kim, B.; Hartman, M. L.; Joseph, L.; Shirihai, O. S.; Vita, J. A. Circulation 2011,
124, 444.
5. Nisoli, E.; Clementi, E.; Paolucci, C.; Cozzi, V.; Tonello, C.; Sciorati, C.; Bracale, R.;
Valerio, A.; Francolini, M.; Moncada, S.; Carruba, M. O. Science 2003, 299, 896.
6. Lira, V. A.; Brown, D. L.; Lira, A. K.; Kavazis, A. N.; Soltow, Q. A.; Zeanah, E. H.;
Criswell, D. S. J. Physiol. 2010, 588, 3551.
7. Tengan, C. H.; Kiyomoto, B. H.; Godinho, R. O.; Gamba, J.; Neves, A. C.; Schmidt,
B.; Oliveira, A. S.; Gabbai, A. A. Biochem. Biophys. Res. Commun. 2007, 359, 771.
8. Nisoli, E.; Falcone, S.; Tonello, C.; Cozzi, V.; Palomba, L.; Fiorani, M.; Pisconti, A.;
Brunelli, S.; Cardile, A.; Francolini, M.; Cantoni, O.; Carruba, M. O.; Moncada, S.;
Clementi, E. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16507.
9. Yamazaki, K. G.; Romero-Perez, D.; Barraza-Hidalgo, M.; Cruz, M.; Rivas, M.;
Cortez-Gomez, B.; Ceballos, G.; Villarreal, F. Am. J. Physiol. Heart Circ. Physiol.
2008, 295, H761.
34. Brinckmann, A.; Weiss, C.; Wilbert, F.; von Moers, A.; Zwirner, A.; Stoltenburg-
Didinger, G.; Wilichowski, E.; Schuelke, M. PLoS ONE 2010, 5, e13513.
35. John, G. B.; Shang, Y.; Li, L.; Renken, C.; Mannella, C. A.; Selker, J. M.; Rangell, L.;
Bennett, M. J.; Zha, J. Mol. Biol. Cell 2005, 16, 1543.
36. Fernie, A. R.; Carrari, F.; Sweetlove, L. J. Curr. Opin. Plant Biol. 2004, 7, 254.
37. Rutter, J.; Winge, D. R.; Schiffman, J. D. Mitochondrion 2010, 10, 393.
38. Scarpulla, R. C. Phys. Rev. 2008, 88, 611.
10. Yamazaki, K. G.; Taub, P. R.; Barraza-Hidalgo, M.; Rivas, M. M.; Zambon, A. C.;
Ceballos, G.; Villarreal, F. J. J. Am. Coll. Cardiol. 2010, 55, 2869.