1 Introduction
NO produced by endothelial cells and stimulating production of cGMP in
the underlying smooth muscle cells, is repeatedly proposed to mediate
endothelium-dependent vasodilatation(Monica, Bian & Murad, 2016;
Shimokawa & Godo, 2020; Vanhoutte, Shimokawa, Feletou & Tang, 2017;
Vanhoutte, Zhao, Xu & Leung, 2016). Superoxide anions
(O2-) which rapidly bind and
inactivate NO, can then cause endothelial dysfunction during conditions
of increased oxidative stress that take part in the pathogenesis of
cardiovascular diseases (CVD)(Daiber et al., 2017; Forstermann &
Munzel, 2006; Zhang, Murugesan, Huang & Cai, 2020). Reported
experimental evidence for these widely accepted mechanisms of
vasodilatation is frequently incomplete and does not exclude alternative
mechanisms. In addition to de novo synthesis, endothelial NO can be
released from intracellular stores and can relax vascular smooth muscle
independently from cGMP(Batenburg, Garrelds, van Kats, Saxena & Danser,
2004; Batenburg et al., 2004). NO-synthases (especially the nNOS
isoform) produce not only NO but also nitroxyl(Fukuto, 2019; Irvine,
Ritchie, Favaloro, Andrews, Widdop & Kemp-Harper, 2008; Schmidt,
Hofmann, Schindler, Shutenko, Cunningham & Feelisch, 1996; Toda &
Okamura, 2003). Like NO, HNO is a potent stimulator of soluble guanylyl
cyclase (sGC) but resists inactivation by
O2-(Arcaro, Lembo & Tocchetti, 2014;
Irvine, Ritchie, Favaloro, Andrews, Widdop & Kemp-Harper, 2008; Leo,
Joshi, Hart & Woodman, 2012). In small muscular resistance-sized
arteries, the relation between oxidative stress in CVD and
endothelium-dependent vasodilatation is even more complex than in large
elastic conduit arteries(Ellinsworth, Sandow, Shukla, Liu, Jeremy &
Gutterman, 2016; Shimokawa & Morikawa, 2005; Vanhoutte, Shimokawa,
Feletou & Tang, 2017). These micro-arteries which control blood
pressure and local blood flow, continue to display endothelium-dependent
vasodilatation when NOS or sGC are inhibited. In human resistance
arteries, this additional pathway may involve
H2O2 produced by the microvascular
endothelial cells and causing hyperpolarization of the underlying smooth
muscle(Ellinsworth, Sandow, Shukla, Liu, Jeremy & Gutterman, 2016;
Leurgans et al., 2016; Shimokawa & Morikawa, 2005; Vanhoutte,
Shimokawa, Feletou & Tang, 2017). Dismutation of
O2- produced by mitochondria, NOS or
NADPH oxidases is the main source of this endothelium-derived
hyperpolarizing H2O2(Shimokawa & Godo,
2020; Widlansky & Gutterman, 2011; Zhang, Murugesan, Huang & Cai,
2020). In human resistance arteries, increased oxidative stress can thus
blunt actions of endothelium-derived NO and promote
endothelium-dependent hyperpolarization. The situation is unclear in
resistance arteries from patients with resistant CVD requiring surgery.
Here, chronic elevation of oxidative stress resulting from multiple risk
factors and a long history of CVD might impair the NOS/NO/sGC pathway
and promote endothelium-dependent hyperpolarization while chronic
treatment with cholesterol lowering, anti-hypertensive and anti-diabetic
drugs that have direct and indirect antioxidant properties(Daiber et
al., 2017), can protect endothelium-derived NO. Recent observations by
Gutterman et al. on endothelium-dependent flow-induced dilatations of
human resistance arteries are partly in line with this proposal. In
contrast to vessels from patients without clinical signs of coronary
artery disease, these dilatations were not inhibited by the non-specific
inhibitor of NO-synthases L-NAME but were blocked by scavenging of
H2O2 with catalase in vessels isolated
from patients undergoing coronary artery bypass grafting(Freed, Beyer,
LoGiudice, Hockenberry & Gutterman, 2014; Schulz, Katunaric,
Hockenberry, Gutterman & Freed, 2019; Zinkevich, Fancher, Gutterman &
Phillips, 2017).
In this study, we tested the hypotheses that
endothelium-dependent
vasodilatation of resistance arteries from patients with resistant CVD
uses mechanisms that are either insensitive to oxidative stress or
involve a reactive oxygen species. For this purpose, we isolated
resistance arteries from biopsies of parietal pericardium obtained
during elective cardiothoracic surgeries. In view of the small size of
the arterial tissue samples, we used immunohistochemistry and
pharmacological tools to demonstrate presence of mechanistic components
and their contribution to the in vitro relaxing effects of the
endothelium-dependent vasodilator bradykinin.