4 Discussion
The main findings of this study are that BK-induced relaxations of
resistance arteries from patients with resistant CVD, are not modified
by c-PTIO, NAC or catalase and only slightly reduced by DETCA. They
suggest that extracellular NO, HNO and
H2O2 do not mediate communication from
endothelium to smooth muscle during endothelium-dependent relaxation of
resistance arteries from these patients and that these relaxations can
proceed during excessive oxidative stress.
Endothelium-dependent vasodilatation of human resistance arteries are
mediated by endothelium-derived prostacyclin, NOS and
endothelium-dependent hyperpolarization in children, adults and patients
with coronary artery disease, respectively(Beyer et al., 2017). Several
laboratories including our own, reported that
H2O2 can act as an endothelium-derived
hyperpolarizing factor in resistance arteries from patients(Beyer et
al., 2017; Ellinsworth, Sandow, Shukla, Liu, Jeremy & Gutterman, 2016;
Leurgans et al., 2016; Schulz, Katunaric, Hockenberry, Gutterman &
Freed, 2019; Shimokawa & Godo, 2020; Shimokawa & Morikawa, 2005).
Relations between this factor, NOS and oxidative stress are
controversial and were the focus of our study. The functional importance
of endothelium-dependent hyperpolarization is largely unknown because it
is usually investigated during pharmacological inhibition of
NO-synthases. In large elastic conduit arteries, endothelial NOS
produces NO that causes smooth muscle relaxation via production of cGMP.
Superoxide anions produced by NADPH oxidases and mitochondria inhibit
this pathway by uncoupling of eNOS, binding and inactivation of NO and
damaging sGC(Daiber et al., 2017; Elbatreek et al., 2020; Evgenov,
Pacher, Schmidt, Hasko, Schmidt & Stasch, 2006; Forstermann & Munzel,
2006; Monica, Bian & Murad, 2016; Zhang, Murugesan, Huang & Cai,
2020). In addition to NO, NOS can produce HNO and
O2-(Forstermann
& Munzel, 2006; Schmidt, Hofmann, Schindler, Shutenko, Cunningham &
Feelisch, 1996). Nitroxyl is not inactivated by
O2- and potently stimulates
sGC(Fukuto, 2019; Irvine, Ritchie, Favaloro, Andrews, Widdop &
Kemp-Harper, 2008; Leo, Joshi, Hart & Woodman, 2012; Toda & Okamura,
2003). Although L-NAME partly inhibits production of
O2- by endothelial NOS(Kaesemeyer,
Ogonowski, Jin, Caldwell & Caldwell, 2000), superoxide synthesized by
NOS is transformed by SOD1 into H2O2 as
an endothelium-derived hyperpolarizing factor in mouse mesenteric
resistance arteries(Morikawa et al., 2003; Takaki et al., 2008). In
coronary arterioles from patients with coronary artery disease, on the
other hand, mitochondria rather than NOS are the source of vasodilator
endothelium-derived H2O2(Beyer et al.,
2017; Freed, Beyer, LoGiudice, Hockenberry & Gutterman, 2014; Schulz,
Katunaric, Hockenberry, Gutterman & Freed, 2019).
With the aim to analyze clinically relevant relations between
endothelium-dependent vasodilatation and oxidative mechanisms, we tested
the hypotheses that BK-induced relaxation of resistance arteries from
patients with resistant CVD is mediated by mechanisms that are either
insensitive to oxidative stress or involve a reactive oxygen species.
For this purpose, we studied resistance artery biopsies obtained during
elective cardiothoracic surgery. The demographic, clinical and vascular
pharmacological properties of the study group varied (Table 1 and Figure
1). Surgery was required despite normalization of classical risk
factors, suggesting future benefit from novel pharmacotherapy inspired
by proven pathogenic mechanisms. Resistance artery contractile and
relaxing responses varied considerably between individual patients
(Figure 1). The different size of the vessels, which is influenced by
sampling bias, contributed little to this inter-assay variability.
Because some of the results were unexpected, we had to confirm the
endothelium-dependence of BK-induced relaxing responses in patient
pericardial resistance arteries(Leurgans et al., 2016).
Immunohistochemical staining of potential key players also displayed
considerable inter-individual variability despite careful titration of
the concentration of the primary antibodies. But, presence of all three
isoforms of NOS, SOD1 and catalase could be demonstrated in the
microvascular wall.
To probe involvement of mediators in relaxing responses we tested
effects of exogenously applied candidate mediators and of potential
scavengers thereof. We confirmed that SNP, a NO-donor, and exogenous
H2O2cause relaxation in patient pericardial resistance arteries(Leurgans et
al., 2016; Leurgans, Bloksgaard, Irmukhamedov, Riber & De Mey, 2018).
We found that CXL-1020, an HNO-donor compound(Arcaro, Lembo &
Tocchetti, 2014), relaxes patient resistance arteries. Exogenous
catalase prevented relaxing effects of exogenous
H2O2, as expected. c-PTIO, ODQ and DETCA
abolished relaxing effects of SNP confirming that they are due to NO
stimulating sGC and sensitive to inactivation by
O2-(Forstermann
& Munzel, 2006; Ignarro et al., 1980; Omar, Cherry, Mortelliti,
Burke-Wolin & Wolin, 1991; Vanhoutte, Zhao, Xu & Leung, 2016). NAC did
not modify responses to SNP but reduced the relaxing potency of CXL-1020
which was not altered by the SOD-inhibitor DETCA. These properties are
very similar to those of nitrergic nervous dilator mechanisms in various
organs that are mediated by HNO generated by neuronal NOS, insensitive
to scavenging and inactivation by endogenous
O2- or c-PTIO but sensitive to
inhibition by NAC(Arcaro, Lembo & Tocchetti, 2014; Fukuto, 2019;
Irvine, Ritchie, Favaloro, Andrews, Widdop & Kemp-Harper, 2008;
Schmidt, Hofmann, Schindler, Shutenko, Cunningham & Feelisch, 1996;
Toda & Okamura, 2003). Apart from exogenous catalase, the actions of
the pharmacological tools that we revalidated are not restricted to the
extracellular space.
DETCA abolished relaxing responses to exogenous NO during
K+-induced contraction and amitrole reduced these
contractions. This suggests a large production of endogenous
O2-leading via dismutation to H2O2 that is
then decomposed by endogenous catalase. This endogenous antioxidant
pathway seems efficacious because addition of exogenous catalase did not
modify contractile responses. No signs of basal production of relaxing
concentrations of HNO were observed since NAC, 7-NI and NPLA did not
modify K+-induced contractions. In contrast to the
selective inhibitors of nNOS, L-NAME increased contractile responses.
This finding has frequently been interpreted as indicative of basal
production of NO. However, c-PTIO and DETCA did not increase contractile
responses and the effect of ODQ did not reach statistical significance
(Figure 6A). Non-canonical effects of L-NAME that are not due to
inhibition of NOS activity have been observed after chronic but not
acute administration of the compound(Kopincova, Puzserova & Bernatova,
2012; Liu et al., 2019). Our findings therefore raise the possibility
that, at least in resistance arteries from patients with CVD, NOS can
generate a dilator compound that is distinct from NO, HNO and
O2-.
We studied agonist-stimulated endothelium-dependent relaxation during i)
contraction stimulated by K+-induced depolarization
that mimics myogenic tone and inhibits hyperpolarizing influences and
during ii) contraction stimulated with ET-1. This stimulator of NADPH
oxidases is upregulated in several CVD (Barton & Yanagisawa, 2019;
Davenport et al., 2016). In patient pericardial resistance arteries, its
vasoconstrictor effect is mediated by ETA receptors and
not modified by direct or endothelium-dependent ETBeffects(Leurgans et al., 2016). We confirmed that in these vessels,
BK-induced relaxation of ET-1 stimulated contraction is larger and
refractory to inhibition by L-NAME and ODQ compared to
K+-induced contraction(Leurgans et al., 2016). Our
earlier proposals on involvement of endothelium-derived NO during
depolarization-induced contraction and of endogenous
H2O2 as an endothelium-derived
hyperpolarizing factor in the presence of ET-1, could however not be
confirmed.
In depolarized arteries, the endothelium-dependent effects of BK were on
average markedly reduced by L-NAME and ODQ. Unlike those of SNP, they
were not modified by c-PTIO and only partly reduced by DETCA. These
properties could suggest involvement of HNO instead of NO. Although the
nitroxyl donor CXL-1020 caused relaxation with an efficacy comparable to
that of BK, effects of the former but not the latter were, however,
reduced by NAC. An involvement of extracellular
H2O2 is equally unlikely. In theory,
H2O2 can be generated by dismutation of
O2- produced by NOS(Morikawa et al.,
2003) and relaxes K+-induced contraction with low
potency. However, BK-induced relaxation of depolarized arteries was only
partly reduced by DETCA and not modified by even a high concentration of
exogenous catalase. Rather, a NOS-derived dilator compound that
stimulates sGC, is distinct from NO and HNO, and resists inactivation by
O2- seems to be involved. This
proposal for agonist-stimulated endothelium-dependent relaxation of
depolarized patient pericardial arteries is comparable to the one
described above for basal conditions. Observations with indomethacin
suggest that such a mechanism is responsible for three quarters of the
relaxation and a dilator prostaglandin for the remaining quarter.
Relaxing responses to BK in arteries contracted with ET-1 were on
average and in contrast to depolarized segments, not modified by L-NAME
or ODQ in the absence and presence of indomethacin. They were shifted to
endothelium-dependent hyperpolarizing responses that could not be
attenuated by scavengers of NO or HNO. This shift is selectively induced
by ET-1 because in our previous study of CVD patient pericardial
resistance arteries, BK-induced relaxation of contractile responses to
the TXA2-analogue U46618, but not ET-1, were
significantly attenuated by L-NAME and indomethacin(Leurgans et al.,
2016). H2O2 did not play a major role
because inhibition of SOD, which is an important albeit not exclusive
source of the peroxide, had only a small effect and especially because
exogenous catalase did not reduce the potency or efficacy of BK in the
presence of ET-1. This seemingly contrasts with previous reports from
several laboratories including our own(Beyer et al., 2017; Ellinsworth,
Sandow, Shukla, Liu, Jeremy & Gutterman, 2016; Freed, Beyer, LoGiudice,
Hockenberry & Gutterman, 2014; Leurgans et al., 2016; Matoba et al.,
2000; Munoz et al., 2018; Schulz, Katunaric, Hockenberry, Gutterman &
Freed, 2019; Shimokawa & Morikawa, 2005). We previously evaluated the
effect of exogenous catalase during simultaneous inhibition of NOS, sGC,
COX and endothelial Ca2+-activated
K+-channels(Leurgans et al., 2016). Almost by
definition, continuous presence of both L-NAME and indomethacin has been
part of most studies of endothelium-dependent hyperpolarization and
evaluations of involvement of endothelium-derived
H2O2 using catalase. Indomethacin was
absent in our present experiments. It may be of interest to analyze in
future studies how cyclooxygenase products modulate the role of
H2O2 as an endothelium-derived
hyperpolarizing factor in the microvascular wall. Earlier conclusions
were strengthened by us and others with imaging studies using dyes that
were designed to demonstrate reactive oxygen species within cells and
not in the extracellular space. In line with this, PEGylated-catalase is
used in recent investigations of H2O2 in
flow-induced endothelium-dependent dilatation of coronary and
subcutaneous arterioles from patients with and without coronary artery
disease(Beyer et al., 2017; Ellinsworth, Sandow, Shukla, Liu, Jeremy &
Gutterman, 2016; Schulz, Katunaric, Hockenberry, Gutterman & Freed,
2019). Several other mechanisms have been proposed to mediate
endothelium-dependent vasodilatation in addition to endothelium-derived
prostaglandins, NO, HNO and
H2O2(Ellinsworth, Sandow, Shukla, Liu,
Jeremy & Gutterman, 2016; Shimokawa & Godo, 2020; Vanhoutte,
Shimokawa, Feletou & Tang, 2017). These include alternative chemical
mediators and electrical communication via heterocellular gap junctions.
Although we did not investigate the latter, it can not explain that
endothelium-dependent relaxation of depolarization-induced contraction
could be inhibited by L-NAME and ODQ but not by a scavenger of NO or by
an inhibitor of SOD.
To better define the molecular mechanisms underlying the observed
profiles of endothelium-dependent vasodilatation, indirect
pharmacological analyses of small number of vessels from the type of
patients that we investigated will not suffice. The patients and their
vessels varied considerably in several aspects. For instance, not only
the amplitudes of the contractions and of the endothelium-dependent
relaxations (Figure 1) but also the extent to which BK-induced
relaxation could be inhibited by L-NAME (Figure 10), varied considerably
between individual patients. As a result, a large number of experiments
was needed to detect for instance i) a significant difference of the
maximal relaxation between patients with and without coronary artery
disease and ii) that the relaxing response in the presence of ET-1 was
not significantly modified by L-NAME. Experimental animal research is
here not an immediate or straightforward solution. To the best of our
knowledge, no type of vessel from an animal model has been described
with comparable vascular pharmacological properties. Future studies can
focus on human resistance arteries that are more widely available and
that can be harvested from groups of individuals with or without
cardiovascular and non-cardiovascular disease. To this end, human
subcutaneous and omental resistance arteries can be considered(Beyer et
al., 2017; Munoz et al., 2018; Shimokawa & Morikawa, 2005; Zinkevich,
Fancher, Gutterman & Phillips, 2017). Pharmacological experiments using
a standardized protocol in vessels from many diverse individuals, can be
of value provided that these individuals are characterized in detail in
terms of not only their demographics and standard risk factors. Large
numbers can form the basis of association studies. Selection of novel
circulating factors can be hypothesis-driven and lead to confirmatory
studies in dedicated experimental animals. These approaches were
recently applied with respect to the role of a subtype of NADPH oxidases
(NOX5) in the development of regional endothelial dysfunction and
systolic hypertension with ageing(Elbatreek et al., 2020).
In resistance arteries from patients with CVD requiring surgery, we
confirmed that endothelium-dependent relaxation can be inhibited by
L-NAME and ODQ during depolarization-induced contraction but not in the
presence of ET-1. A role for NO, HNO or
H2O2 in these endothelium-dependent
relaxations could not be confirmed. Alternative mechanisms seem to be
involved. They do not depend on reactive oxygen species and are
resistant to elevated levels of superoxide anions.