Báo cáo khoa học: Pharmacology of vascular endothelium Delivered on 27 June 2004 at the 29th FEBS Congress in Warsaw pptx

THE SIR HANS KREBS LECTURE
Pharmacology of vascular endothelium
Delivered on 27 June 2004 at the 29th FEBS Congress in Warsaw
Ryszard J. Gryglewski
Jagiellonian University, Cracow, Poland
Sir Hans Krebs was one of the most versatile biochem-
ists of the twentieth century. Many of his sayings stay
as bright as his science. My favourite quotation is: ‘‘…
we all are committed to correlating biochemical events
to function … the point I want to make is that it is not
always immediately clear what their relevance to func-
tion may be …’’ [1]. Indeed, a burning desire for imme-
diate comprehension, amplified by the abomination of
being just another fact collector may overcome rational
cautiousness. We pharmacologists know this only too
well. Sir John Vane urged his young followers: ‘‘Do
simple experiments and make simple hypotheses – there
are plenty of others who will come along and show
how much more complicated the answer really is …’’
[2]. Keeping in mind the above advice, I present the
vascular endothelium as a newly discovered target for
the pharmacotherapy of arterial hypertension, athero-
thrombosis and diabetic angiopathies.
I intend to focus on the pharmacology of the
endothelial prostacyclin ⁄ nitric oxide radical (PGI
2
⁄
Keywords
ACE-I; ASA; bradykinin; endothelial
dysfunction; nitric oxide; prostacyclin;
statins; thienopyridines

Correspondence
R. J. Gryglewski, Jagiellonian University,
Kasztelan˜ ska 30, 30-116 Cracow, Poland
E-mail:
(Received 10 February 2005, revised
13 April 2005, accepted 20 April 2005)
doi:10.1111/j.1742-4658.2005.04725.x
Sir John Vane named vascular endothelium ‘the maestro of blood circula-
tion’. Recently, ‘the maestro’ has become a target for pharmacotherapy of
atherothrombotic and diabetic vasculopathies with well known cardio-
vascular drugs belonging to the families of Angiotensin Converting Enzyme
inhibitors, HMG CoA reductase inhibitors or b
1
-Adrenoceptor antagonists.
These drugs became upgraded to a position of the pleiotropic endothelial
drugs. It is not a simple verbal change in the nomenclature. It means that
these drugs apart from their well defined mechanisms of action, as indi-
cated in their regular names, in addition they act in an unknown mechan-
ism at the level of vascular endothelium preventing angina, myocardial
infarction and stroke. Many biochemical events take place in endothelial
cells. I chose for a closer inspection the nitric oxide/prostacyclin defensive
system to explain the endothelial pleiotropism of the drugs in question. I
tried to examine the validity of this conception according to the general
rule: in vitro cognitio sed in vivo veritas.
Abbreviations
AA, arachidonic acid; ACE-I, angiotensin converting enzyme (and kininase 2) inhibitors; ADMA, asymmetric dimethylarginine; ASA,
acetylsalicylic acid; BH4, tetrahydrobiopterin; Bk, bradykinin; BPF, bradykinin potentiating factor; CAD, coronary heart disease; CaM,
calmodulin; COX-1, constitutive cyclooxygenase 1; COX-2, inducible cyclooxygenase 2; EDHF, endothelium-derived hyperpolarizing factor;
EDRF, endothelium-derived relaxing factor; EETs, cis-epoxyeicosatrienoic acids; eNOS, constitutive endothelial nitric oxide synthase; FAD,
flavin adenine dinucleotide; FMD, flow mediated dilatation (of brachial artery in humans); FMN, flavin mononucleotide; HMG-CoA,

hydroxymethylglutaryl coenzyme A; HO-1, inducible heme oxygenase; 15-HPAA, 15-hydroperoxyarachidonic acid; HYHC, hyperhomo-
cysteinemia; 6-keto-PGF
1a
, prostaglandin 6-keto-PGF
1a
, a stable product of decomposition of PGI
2
; LDL, low-density lipoproteins; L-NAME,
L-N(G)-nitroarginine methyl ester, a nonselective NOS inhibitor; NOHA, N
’
-hydroxy-Arg; ONOO
–
, peroxynitrite; ox-LDL, oxidized low-density
lipoproteins; PARP, poly ADP ribosyl polymerase; PGE
2
, prostaglandin E
2
; PGHS2, PGH
2
synthase; PGI
2
, prostacyclin; PGIS, prostacyclin
synthase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDMA, symmetric dimethylarginine; TXA
2
, thromboxane A
2
;
TXAS, thromboxane A
2
synthase; TXB

2
, thromboxane B
2
.
2956 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS
NO
•
) defence system. Other aspects of endothelial
biology are reviewed by Nachman and Jaffe [3] with
a special attention being paid to the functioning of
Weibel–Palade bodies and their response to proin-
flammatory or prothrombotic agents as manifested by
the release of von Willebrand factor, P selectin and
interleukin-8. Readers interested in the endothelial
mitochondrion as a propagator of oxidative stress [4]
and the mitochondrion-oriented role of reactive oxy-
gen species (ROS) and hydrogen peroxide [5] are
directed to studies by Keaney and coworkers [4,5].
Mitochondrial oxidases along with NAD(P)H oxidase,
xanthine oxidase and uncoupled constitutive endothel-
ial nitric oxide synthase (eNOS) constitute the source
of endothelial ROS, which may act as modulators of
tone, growth and remodelling of the vascular wall. It
may well be that inflammation plays a primary role
in atherogenesis, whereas oxidative stress is a secon-
dary phenomenon [6]. At low concentrations, ROS
may protect endothelial cells against apoptotic beha-
viour [7]. Long-term treatment with antioxidant vita-
mins does not influence the course of the disease
or correct endothelial dysfunction in patients with

atherosclerosis [8]. The great expectations for the
therapeutic use of antioxidants in patients with athero-
sclerosis need to be re-examined.
Endothelium as the endocrine organ
Why does blood not coagulate within healthy blood
vessels? This question has been addressed for centuries.
The warmth of the body (Plato), the lack of contact
with air (James Hewson) and the vital power of blood
(John Hunter) have all been claimed as reasons. The
truth is that vascular endothelium secretes a bunch of
antithrombotic and thrombolytic mediators that keep
blood fluid within an undamaged circulatory system.
Vascular endothelium is neither a ‘primitive mem-
brane’, as claimed by Rudolph von Virchow, nor a
‘nucleated sheet of cellophane’, as Sir Howard Florey
stated [9]. Sir John Vane named the endothelium ‘the
maestro of blood circulation’ [10], which should be
viewed as a peculiar dissipated endocrine organ (mass
 1000 g, surface area  100 m
2
). Among others sub-
stances, endothelium releases into the passing blood –
labile, lipophilic and antithrombotic local hormones
like PGI
2
and NO
•
as well as a peptide – tissue plasmi-
nogen activator. These prevent the build up of thrombi
and disperse any thrombi at an early stage of their for-

mation. This is why blood stays fluid within a healthy
vascular bed. The inherent chemical instability of PGI
2
and NO
•
allows for the immediate transformation of
extravasated blood into a haemostatic plug. Unfortu-
nately, the same transformation may occur locally
inside the circulatory system of patients with athero-
sclerotic plaques or diabetic angiopathies. The endo-
thelium then loses its protective properties and may
even produce proinflammatory and thrombogenic
agents (endothelial dysfunction).
Endothelium generates many biologically active sub-
stances other than PGI
2
or NO
•
, to mention just
four regioisomers of cis-epoxyeicosatrienoic acid (EETs)
produced from AA by CYP2J2 epoxygenases [11].
EETs are vasoprotective vasodilators. Some may be
responsible for the activity of endothelium-derived
hyperpolarizing factor (EDHF) [11], and for prevent-
ing platelet adhesion to endothelium [12]. A potent
vasoconstrictor, endothelin, is also produced [13], as
are a vast number of mediators of haemostasis, growth
factors and cytokines [14]. The outer endothelial layer
of the glycocalyx houses the membrane sensors for
shear stress and various types of endothelial receptors

such as B
2
for bradykinin (Bk), P
2y
subtypes for ADP
from platelets and ATP from erythrocytes, PAF-R for
platelet activating factor (PAF) from leukocytes, and
PAR for thrombin [15]. The membrane-bound endo-
thelial enzymes include kininase 2, also called angio-
tensin 1-converting enzyme (ACE-I).
Prostacyclin
Prostacyclin (PGI
2
) was discovered in 1976 during the
search for biological systems that in addition to blood
platelets might convert prostaglandin endoperoxides
(PGG
2
or PGH
2
) to thromboxane A
2
(TXA
2
) [16,17].
This search was possible because newly discovered
PGG
2
and PGH
2

were kindly offered to John R. Vane
by the discoverer of TXA
2
, Bengt Samuelsson of the
Karolinska Institutet. This search was not successful,
except for the detection of minute amounts of TXA
2
made from PGH
2
by lung and spleen microsomes.
Instead we found that a microsomal fraction of pig
aorta transformed prostaglandin endoperoxides into
an unknown, unstable substance (with a half-life of
4 min at 37 °C) that had vasodilator and platelet-
suppressant properties in vitro. This substance was
later named prostacyclin (PGI
2
). Further studies
revealed that PGI
2
, when administered intravenously,
dissipated platelet-rich thrombi in arterial blood in vivo
[18] and that this effect was augmented by theophyl-
line. This latter finding confirmed that a cyclic nucleo-
tide (in this case cAMP) was the second messenger of
PGI
2
in platelets [19].
The common precursor for prostanoids including
PGI

2
is the four double-bonds 2-carbon fatty acid –
arachidonic acid (AA). It was found that the nonenzy-
R. J. Gryglewski Endothelium and drugs
FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2957
matic product of AA monooxygenation (15-hydro-
peroxy-arachidonic acid; 15-HPAA) and other linear
lipid peroxides are inhibitors of microsomal prosta-
cyclin synthase (PGIS). Therefore, we hypothesized
that PGI
2
deficiency resulted from an excessive non-
enzymatic peroxidation of body lipids might contribute
to development of atherosclerosis [20]. Consequently,
we hoped to use synthetic PGI
2
as a replacement ther-
apy in patients with atherosclerosis.
Actually, I was the first healthy volunteer to receive
an intravenous infusion of synthetic PGI
2
sodium salt.
I lost the noble position of an observer during the last
stage of this experiment. Still, these early trials allowed
us to establish a range of therapeutic doses for PGI
2
and to observe the side effects caused by its overdos-
age [21]. Eventually, PGI
2
was infused into patients

with atherosclerosis of the leg arteries [22]. However,
like most other powerful biological mediators, both
PGI
2
(epoprostenol) [23] and its stable analogues (e.g.
iloprost) [24] never became first-line drugs for the
treatment of atherothrombosis, instead giving way to
drugs that act as releasers of endogenous endothelial
PGI
2
[25]. However, some PGI
2
analogues (e.g. tre-
prostinil) are still used to treat patients with pulmon-
ary arterial hypertension [26], including those with
connective tissue disease [27].
The lung is a rich source of eicosanoids including
leukotrienes. Various prostanoids are generated within
different pulmonary compartments. Tracheal smooth
muscles generate PGE
2
, contractile elements of lung
parenchyma generate TXA
2
[28], whereas pulmonary
endothelium secrets PGI
2
. We hypothesized [29] that
pulmonary endothelium may serve as a source for
circulating PGI

2
[30]. This concept was not well
accepted. What kind of a circulating hormone has a
half-life in blood of 3–4 min? Nonetheless, assuming
PGI
2
is generated continuously by pulmonary endo-
thelium, the stability of PGI
2
might be sufficient for
it to be transported within the blood from the lung
to atherosclerotic coronary or cerebral arteries with
dysfunctional endothelium, and to save them from
being occluded by platelet-rich thrombi. Pulmonary
endothelium might be a good target for new specific
releasers of circulating PGI
2
, although the local gen-
eration of PGI
2
by the endothelium lining the vascu-
lar tree is probably a more important therapeutic
target, at least up to the point when the efficacy of
peripheral endothelium in not seriously disturbed by
an advanced atherothrombosis. Interestingly, overex-
pression of pulmonary PGIS decreases the incidence
of cancerogenesis in murine models of lung cancer [31].
The crude microsomal fraction of aortic homogen-
ates that allowed us to discover biosynthesis the of
PGI

2
from PGH
2
[16,17] contained PGIS. This enzyme
was purified and characterized as a member of cyto-
chrome P450 family (CYP 8A1) [32]. In endothelial
cells it collaborates with a supplier of PGH
2
, i.e. with
PGH
2
synthase (PGHS-2), commonly, but less pre-
cisely, called cyclooxygenase 2 (COX-2). In endothelial
cells COX-2 is induced by shear stress. COX-2 seems
to be the major source of systemic PGI
2
in healthy
humans [33]. In female mice oestrogens upregulate
PGI
2
production via COX-2, and subsequently offer
protection against atherothrombosis [34]. Also, intra-
vascular thrombosis in rats next to hypoxia-induced
hypertension is prevented by the upregulation of
vascular COX-2 followed by increased generation of
PGI
2
[35].
There is little doubt that, in humans and laboratory
animals, the endothelial COX-2 ⁄ PGIS tandem is

responsible for the generation of vasoprotective PGI
2
,
whereas in blood platelets the constitutive cyclooxy-
genase 1 ⁄ thromboxane A
2
synthase (COX-1 ⁄ TXAS)
tandem generates vasotoxic TXA
2
.
Nitric oxide radical
In 1980, a series of in vitro experiments with acetyl-
choline-treated aortic rings led Robert Furchgott to
discover endothelium-derived relaxing factor (EDRF)
[36]. Robert Furchgott likes to say that his great dis-
covery arose from a number of accidental findings.
Those in 1986 exploded in the grand finale, i.e. in the
discovery that EDRF is nitric oxide. Actually, the idea
that EDRF ¼ NO was proposed by Robert Furchgott
and Louis Ignarro, independently [37]. Robert Furchg-
ott is modest as only a great scholar can be. His mod-
esty provokes the quotation from Louis Pasteur: ‘‘…
where observation is concerned, chance favours only
the prepared mind’’.
The fabulous story of the discovery of EDRF(NO)
was presented by Robert Furchgott [37], Louis Ignarro
[38] and Ferid Murad [39,40] – three 1998 Nobel prize
laureates in medicine and physiology. In vascular endo-
thelium, NO
•

is synthetized from Arg by eNOS, which
competes for substrate with tissue arginases. eNOS is a
homodimeric oxidoreductase with NADPH, flavin
mononucleotide (FMN), flavin adenine dinucleotide
(FAD), calmodulin (CaM) and tetrahydrrobiopterin
(BH4) acting as cofactors. eNOS via N’-hydroxy-Arg
(NOHA) generates NO
•
and citrulline. Physiologically,
eNOS homodimer catalyses a five-electron oxidation of
Arg, whereas BH4 plays a crucial role in the activation
of dioxygen. In tissues, NO
•
is a powerful endogenous
stimulator of soluble cytosolic guanylate cyclase. Thus
made, cGMP is the second messenger for NO
•
in the
Endothelium and drugs R. J. Gryglewski
2958 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS
same way that cAMP is the second messenger for
PGI
2
. Both cyclic nucleotides mediate the vasodilator,
vasoprotective and platelet-suppressant activities of
NO
•
and PGI
2
, respectively.

However, when eNOS splits into monomers, the
eNOS monomer acts as a reductase, and one-electron
reduction of dioxygen leads to formation of a super-
oxide anion (O
2
–
) [41]. Uncoupling of eNOS occurs
as a consequence of BH4 shortage resulting from
folate avitaminosis or from hyperhomocysteinaemia
(HYHC) [42]. Apart from the uncoupling of eNOS,
the other source of vascular O
2
–
might be NAD(P)H
oxidase (43). Dimerization of eNOS requires the
intracellular availability of the substrate, i.e. Arg.
This is ensured by the high-affinity cationic cell mem-
brane transporter for Arg. Its functioning might be
invalidated by homocysteine or by asymmetric dime-
thylarginine (ADMA) (see below). Arg supplementa-
tion in patients with atherosclerosis may be also
desirable because Arg acts as a direct antioxidant. In
addition, Arg promotes the secretion of insulin from
pancreatic b cells and the release of histamine from
mast cells – both being vasodilators. Theoretically,
Arg may also produce unfavourable effects, such as
generation of S-adenosyl-homocysteine from S-adeno-
sylmethionine via the methylation-dependent biosyn-
thesis of creatinine from guanidine acetate (44). Yet,
the net therapeutic effect of Arg given orally to

patients with myocardial infarction is encouraging
(45) pointing to a favourable route of biotransforma-
tion in these patients.
Patrick Valance discovered, in human plasma, the
presence of symmetric dimethylarginine (SDMA) and
ADMA. Only ADMA is biologically active, i.e. it acts
as endogenous inhibitor of eNOS and inhibitor of Arg
membrane transporter. Clinical data on ADMA are
growing. A high plasma level of ADMA is considered
a novel cardiovascular risk factor. Nowadays, it is
clear that ADMA contributes to vascular pathology
in atherothrombotic and diabetic angiopathies, pre-
eclampsia and hypertension (46). Elevated plasma lev-
els of ADMA in those patients may also explain the
‘arginine paradox’, i.e. that therapeutic supplementa-
tion with exogenous Arg is beneficial, although in
these patients plasma levels of endogenous Arg exceed
the Michaelis–Menten constant (K
m
) for purified eNOS
in vitro by 25-fold [47].
Prostacyclin and nitric oxide radicals
A complex relationship exists between these two unsta-
ble, lipophylic endothelial secretagogues. At the time
when NO
•
still was known as EDRF it was claimed
that porcine aorta endothelial cells cultured on cytodex
beads, loaded into a heated column and perfused with
Krebs’ buffer, when stimulated with Bk or calcium

ionophore, released both PGI
2
and EDRF in a cou-
pled manner [48]. Superoxide anions abolished the
biological activity of the released EDRF from these
cultured endothelial cells [49], and from native endo-
thelium of perfused canine artery [50]. These latter
findings initiated a march towards the discovery of the
product of the interaction between NO
•
and O
2
–
, i.e.
peroxynitrite (ONOO
–
). ONOO
–
is one of the most
reactive nitrogen species (RNS). It arises most easily
when the eNOS dimer coexists in the vicinity of a
eNOS monomer – then both genders of labile free rad-
icals, i.e. NO
•
and O
2
–
arise side by side, and without
any delay ONOO
–

is made.
ONOO
–
is a powerful oxidant and nitrating agent
that destroys the ‘macromolecules of life’, i.e. proteins
(e.g. PGIS inactivation), lipids [e.g. the generation of
oxidized low-density lipoprotein (ox-LDL) and iso-
prostanes], and nucleic acids [e.g. DNA strand break-
age with a subsequent activation of poly-ADP ribosyl
polymerase (PARP)] [51].
The toxic properties of ONOO
–
play a major role in
atherothrombotic and diabetic angiopathies. In those
endothelial cells, ONOO
–
oxidizes the four zinc thio-
late centres of dimeric eNOS. As a consequence, zinc
atoms are removed and disulfide monomers of eNOS
arise. The coexistence of dimeric and monomeric forms
of eNOS is responsible for the further amplification of
ONOO
–
generation by endothelial cells. This newly
made ONOO
–
selectively nitrates Tyr430 in the enzy-
mic protein of endothelial PGIS. When PGI
2
is elimin-

ated from the endothelial defence system TXA
2
and
PGH
2
gain the upper hand [52].
Endothelial NOS received the mischievous name of
‘the Cinderella of inflammation’ [53]. The authors had
in mind that excessive stimulation of eNOS might lead
to increased vascular permeability by NO
•
, and thus
to inflammation. However, in light of the foul games
played between homodimeric and monomeric forms of
eNOS, ending with the generation of ONOO
–
which
eliminates PGI
2
– the best friend of NO
•
– I would
rather think of eNOS as ‘the Lady Macbeth of athero-
thrombosis’.
Endothelial pharmacology
Samuel Beckett (1906–1989) wrote: ‘‘we need new par-
adigms to accommodate the mess’. The paradigm of
‘pleiotropic action’ for some of cardiovascular drugs
was coined to accommodate a discrepancy between
their officially accepted modes of action and their

R. J. Gryglewski Endothelium and drugs
FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2959
additional therapeutic properties, as reported unexpect-
edly, but repeatedly, by clinicians. For example, statins
were introduced to the clinic with the aim of lowering
blood levels of low-density lipoprotein (LDL) cho-
lesterol, however, they were also found to correct
symptoms of myocardial and cerebral ischaemia, inde-
pendent of their capacity to inhibit hydroxymethyl-
glutaryl coenzyme A (HMG CoA) reductase [54–56].
Further support for the existence of the ‘pleiotropic
action’ of cardiovascular drugs was offered by the
efficacy of ACE-I to protect against myocardial isch-
aemia, stroke and diabetic angiopathies, as confirmed
in multicentre trials that included over 25 000 patients
[57], whereas the classic indication for ACE-I was the
treatment of patients with arterial hypertension. The
phrase ‘pleiotropic action’ is not a cognitive descrip-
tion of reality. Rather, it is an attempt ‘to accommo-
date the mess’. Our experimental data [58,59] pointed
to the possibility that the pleiotropic action of ACE-I
and statins might be explained by their stimulatory
effect on the endothelial generation of PGI
2
and NO
•
.
There are other propositions concerning the mechan-
ism of endothelial actions of statins, e.g. the induction
of heme oxygenase (HO-1) [60] with a subsequent anti-

oxidant effect of biliverdin and CO mediation. Here, I
take the opportunity to present our conception of the
endothelial pharmacology emerging from clinical
observations on the unexpected therapeutic effects of
known cardiovascular drugs. This conception embraces
not only ACE-I and statins, but also other cardiovas-
cular drugs, e.g. nebivolol and carvedilol (b-adrenergic
receptor antagonists) as well as ticlopidine and clopi-
dogrel (antiplatelet thienopyridines).
In vivo assay of endothelial function
Clinicians have developed an excellent noninvasive
method to measure endothelial function in humans.
The method is based on the ultrasound scanning of
the flow-mediated dilatation (FMD) of the brachial
artery after its occlusion and reopening [61]. In prin-
ciple, the FMD response is proportional to the
amount of NO
•
released from endothelium of the vas-
cular bed in question, however, an additional bio-
chemical assay pointed to the release of PGI
2
, along
with NO
•
, from the endothelium during FMD [62].
No wonder – in vitro cultured endothelial cells
released EDRF(NO) and PGI
2
in a coupled manner

[48]. The FMD method allowed the detection of endo-
thelial dysfunction in patients with arterial hyperten-
sion [62], in patients with atherosclerosis undergoing
percutaneous coronary intervention with stenting [63],
and in patients with type 2 diabetes [64]. In patients
with chest pain, a depressed FMD of the brachial
artery was a sensitive indicator of coronary heart dis-
ease (CAD) [65]. FMD is impaired in tobacco smokers
and in smokeless tobacco users compared with tobacco
nonusers [66]. There is ample evidence for the state-
ment that endothelial dysfunction occurs in patients
with hypertension, atherosclerosis and type 2 diabetes,
as well as in tobacco users.
In vitro cognitio sed in vivo veritas (in vitro one may
look for meaning, however, only in vivo is the truth to
be found). This motto stimulated us to develop our own
experimental model for the in vivo assay of endothelial
function [18–20,29,59,67–70]. In our in vivo method it is
not the vasodilator response (as in the case of FMD in
humans) but rather the thrombolytic response that is
used to assess endothelial capacity. Therefore, it is the
endothelial release of PGI
2
that is appreciated at
the first place, whereas the release of NO
•
remains in the
background. Heparinized cats, rabbits and, most fre-
quently, Wistar rats under general anaesthesia with
extracorporeal circulation are used. The arterial blood

superfuses (2–3 mLÆmin
)1
) a collagen strip attached to a
balance. Blood returns to the venous system. Thrombus
mass is recorded continuously along with arterial blood
pressure (Fig. 1). Platelet-rich thrombus [70] gains a
maximum mass of  100 mg within 30 min and stays
unchanged for at least 4 h, unless a stimulator of vas-
cular endothelium (e.g. Ach, Bk or an endotheliotropic
drug) is injected intravenously. Then thrombolysis
occurs (Fig. 1). Its intensity and duration correlate with
plasma levels of prostaglandin 6-keto-PGF
1a
(6-keto-
PGF
1a
), whereas the levels of other stable prostanoids
do not (Fig. 2). The participation of endothelial NO
•
in
thrombolytic response is checked by the pretreatment of
animals with l-NAME or with any other NOS inhib-
itor. The participation of endogenous bradykinin in this
response was checked by pretreatment with Icatibant,
an antagonist of B2 receptors (Fig. 1A). In this system,
thrombi were dissipated by intravenous administration
of PGI
2
sodium salt or by its stable analogue (e.g. ilo-
prost). NO-donors (glyceryl trinitrate, molsidomine,

sodium nitropusside, NONOates) also produced throm-
bolysis but their effective doses were at a range of three
orders of magnitude higher than those required for
PGI
2
or for its analogues. Unlike PGI
2
, NO-donors at
thrombolytic doses were highly hypotensive.
Angiotensin-converting enzyme
inhibitors
ACE-I, this name does not do justice to this class of
drugs, namely captopril, enalapril, and especially per-
indopril, quinapril, ramipril and many other lipophylic
Endothelium and drugs R. J. Gryglewski
2960 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS
ACE-I. There is no doubt that the pharmacological
activity of ACE-I is associated with the elimination of
cytotoxic and vasoconstrictor angiotensin 2, however,
the endothelial action of those ACE-I is also executed
via the local vascular accumulation of Bk, as our data
clearly show (Fig. 1A) [59,67–69].
In 1965 a young Brazilian researcher, Sergio Ferreira
discovered the ‘bradykinin potentiating factor’ (BPF)
in the venom of Brazilian viper Bothrops jararaca [71].
At John Vane’s laboratory in London (where Sergio
Ferreira was a visitor) his discovery was appreciated
than it should have been. At the time, Bk was per-
ceived as a mediator of pain and inflammation respon-
sible for paralytic vasodilatation in the course of acute

pancreatitis. The reasoning was as follows: BPF might
be good for this particular viper for swift killing of its
victims but for us humans – it is no good at all. So,
why should we care about BPF?
Perfusate from isolated guinea-pig lungs dripping
over Vane’s bioassay cascade was used to study Bk
[71] and angiotensin 1 [72] metabolism. Fortunately, it
was soon found that various fractions of BPF given
via the lungs inhibited the conversion of angiotensin 1
to angiotensin 2, and thus BPF was proved to act also
as an ACE-I [73]. Inhibiting the conversion of biolo-
gically inactive angiotensin 1 to hypertensive angioten-
sin 2 – yes, it was an excellent principle on which to
develop a new class of antihypertensive drugs [74].
Indeed, at the request of John Vane, the top industrial
chemists eventually did [75], and the first orally active
ACE-I (a proline derivative – captopril) was intro-
duced for the treatment of arterial hypertension.
The TREND trial [76] offered the first direct clinical
evidence of improvement, by an ACE-I (quinapril), in
endothelium-dependent vasorelaxation in patients with
CAD. There then appeared a number of clinical trials
pointing to the same mechanism of vascular protection
by various ACE-I in patients at high risk of athero-
thrombotic and diabetic vasculopathies [57].
B
A
BP
THR
THR

Dose-dependent thrombolysis by perindopril µg/kg i.v.
Thrombolysis by QUINAPRIL depends on the release of
endogenous bradykinin and PGl
2
– only partially on NO
THR
THR
THR
30 min
mg
mg
100
0
100
0
THR
rat 1
rat 2
rat 3 rat 4
icatibant 100 µg/kg
indomethacin 5 mg/kg
L-NAME 5 mg/kg
quinapril 30 µg/kg
quinapril 30 µg/kg
quinapril 30 µg/kg
quinapril 30 µg/kg
0
100
mg
Thrombogenesis

thrombus weight
Thrombolysis
pressure transducer
weight
transducer
carotid artery
carotid artery
arterial blood
arterial blood
collagen
collagen
THROMBUS
i.v. drug injection
jugular vein
PERINDOPRIL
30.
10.
3.
30 min
100
mg
0
Fig. 1. In vivo bioassay of endothelial secretory function.
Fig. 2. Effect of quinapril on prostanoid plasma levels in Wistar rats
(n ¼ 7).
R. J. Gryglewski Endothelium and drugs
FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2961
Bk is the most potent releaser of PGI
2
from cultured

endothelial cells [48], and the most potent thrombolytic
agent acting via endothelial B
2
receptors in vivo [67].
In Wistar rats, exogenous Bk at thrombolytic doses
is strongly hypotensive. In contrast, endogenous Bk
released from vascular endothelium by low doses of
ACE-I (quinapril > perindopril > captopril) evokes
thrombolysis, but not a fall in blood pressure [59,68].
The principal mechanism of the thrombolytic action of
ACE-I stems from their secondary nature (or rather
their primary nature) of being BPF [71]. Moreover,
there exist other Bk-potentiating effects of exogenous
ACE-I, such as the upregulation of B2 receptors, the
induction and activation of B1 receptors in the endo-
thelium and the stimulation of biosynthesis of angio-
tensin (1–7), which acts as an endogenous ACE-I (that
is BPF) [77]. It should be added that in cultured endo-
thelial cells Bk acts as a ‘minicytokin’, inducing
mRNA for HO-1 and COX-2 [67]. The interaction
between these two enzyme systems was claimed to
amplify the generation of PGI
2
[78]. It may well be
that, in addition to the immediate thrombolytic effects
of ACE-I, chronic treatment with ACE-I offers an
additional advantage of increasing the efficacy of the
endothelial enzymic raft (COX-2 ⁄ PGIS) responsible
for the biosynthesis of vascular prostacyclin along with
increasing local levels of CO and biliverdin – the

defensive products of endothelial HO-1.
In our in vivo model for studying endothelial-medi-
ated thrombolysis in Wistar rats [59,68,69] it was
found that ACE-I (captopril < perindopril < quina-
pril) at low nonhypotensive intravenous doses of 10–
60 lgÆkg
)1
dissipated thrombi that were superfused
with arterial blood. The intensity and duration of this
thrombolysis were paralleled by an increase in arterial
plasma levels of 6-keto-PGF
1a
, and no change in
plasma levels of TXB
2
and PGE
2
(Figs 1 and 2).
Thrombolysis and prostacyclinaemia by ACE-I were
blunted or abolished by pretreatment with icatibant (a
B2 Bk receptor antagonist), by acetylsalicylic acid
(ASA) at a high dose of 50 mgÆkg
)1
(Fig. 3), and by
the coxibs (rofecoxib > celecoxib > nimesulide) at
low doses of 30–300 lgÆkg
)1
. Thrombolysis by ACE-I
was augmented by pretreatment with ASA at a dose of
1mgÆkg

)1
(Fig. 3) or by acetaminophenen. Pretreat-
ment with l-NAME delayed and flattened the throm-
bolytic response to ACE-I only slightly (Fig. 1).
Pharmacological analysis of the above data led us to
conclude that ACE-I evoked thrombolysis by pre-
venting endothelial Bk from being destroyed by cell
membrane-bound ACE. Bk that appeared at the
endothelial cell surface stimulated B2 receptors, which
triggered the COX-2 ⁄ PGIS system to generate PGI
2
,
and e-NOS to generate NO. The final thrombolytic
response to ACE-I depended mainly on PGI
2
, whereas
NO
•
served as a helper with a permissive action. The
endothelial release of NO
•
did not appear as the
conditio sine qua non for thrombolytic response to
ACE-I (Fig. 1A).
There is another conclusion that derives from these
studies. It is as follows: effective endothelial COX-2
inhibition might be followed by thrombogenesis,
whereas preferential COX-1 inhibition in platelets rein-
forced the vasoprotective action of ACE-I (Fig. 3).
Our data cannot be considered as a good prognostic

for the clinical use of high doses of coxibs in patients
with cardiovascular disorders, but they do support the
idea of administrating of low doses of ASA along with
ACE-I (Fig. 3).
Statins
In our in vivo model statins (e.g. atorvastatin and
simvastatin) produce endothelium-mediated, PGI
2
-
dependent thrombolysis when administered intraven-
ously at doses 2–3 orders of magnitude higher than
those for ACE-I [59]. In Langendorff’s preparation of
guinea-pig heart, statins produce NO
•
-dependent vaso-
dilatation of coronary vascular bed [59]. The precon-
tracted bovine coronary artery rings with endothelium
are relaxed by statins, partially via a NO
•
⁄ PGI
2
-
dependent mechanism [79]. In cultured bovine aortic
endothelial cells lipophylic statins, i.e. atorvastatin,
simvastatin and lovastatin (but not a hydrophilic
pravastatin) at a concentration of 30 lm mobilize free
cytoplasmic calcium [Ca
2+
]
i

to 30–50% of that
induced by Bk at a concentration of 10 nm. In the
case of simvastatin and lovastatin, this effect disap-
pears if their lactone rings are hydrolysed [80]. The
above endotheliotropic properties of statins are hardly
Fig. 3. Dose-dependent effect of aspirin (ASA) on perindopril-
induced thrombolysis.
Endothelium and drugs R. J. Gryglewski
2962 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS
associated with their inhibitory action on HMG CoA
reductase. In genetic and pharmacological models of
rat hypertension, rosuvastatin, another lipophilic sta-
tin, was found to exert a beneficial pleiotropic endo-
thelial effect [81]. Patients with acute coronary
syndromes benefit from statin therapy [82]. Statins
mobilize bone-marrow-derived endothelial progenitor
cells [83] and exert a vast number of other pharmaco-
logical effects that are not associated with modulation
of the lipoprotein profile by statins. These unexpected
effects of statins are generally described as ‘pleiotropic
effects’ [84], and one of them is the endotheliotropic
action of statins described by us [59,79,80]. The mode
of activation of the endothelial PGI
2
⁄ NO
•
system by
statins is not clear. An interesting proposal was put
forward by Bill Sessa [85].
Thienopyridines and some of

b
1
-adrenergic receptor antagonists
Here we present two groups of highly effective cardio-
vascular drugs, the efficiency of which may or may not
depend on their additional stimulatory action of vas-
cular endothelium.
Thienopyridines (ticlopidine and clopidogrel) belong
to a family of antiplatelet drugs, however, in vitro they
do not inhibit platelet aggregation. Their in vivo plate-
let-suppressant action is executed by their labile meta-
bolites. Therefore, a substantial lag period is required
for the appearance of the antiplatelet action of thieno-
pyridines. Only unstable metabolites of theirs are cap-
able of antagonizing endogenous ADP on P2y12
purinergic platelet receptors, which when activated by
ADP induce platelet release and platelet aggregation
[86]. The clopidogrel metabolite exerts its antiplatelet
action at IC
50
¼ 1.8 lm [87]. There exists ample evi-
dence for the high efficacy of thienopyridines (especi-
ally clopidogrel) in the treatment of patients with
advanced atherothrombosis of coronary or cerebral
arteries, to mention only the following megatrials:
clopidogrel vs. aspirin in patients at risk of ischemic
events (CAPRIE) [88], clopidogrel in unstable angina
to prevent recurrent events (CURE) and management
of atherothrombosis with clopidogrel in high risk
patients with recent transient ischaemic attacks or isch-

aemic stroke (MATCH) [89].
In 1996 [90] we demonstrated that ticlopidine
(10 mgÆkg
)1
) given intravenously to cats with extracor-
poreal circulation evoked immediate dissipation of the
platelet-rich clots superfused with their arterial blood
[18]. This thrombolytic effect of ticlopidine was com-
parable with that induced by PGI
2
at 0.3 lgÆkg
)1
.
These and other data [90] prompted us to postulate
that the therapeutic efficacy of ticlopidine might be
associated not only with the delayed platelet-suppres-
sant effect of its unstable metabolite via blockade of
P2y12 platelet receptors, but also with the instan-
taneous endothelial action of the native molecule
of ticlopidine showing up as an immediate, endo-
thelium-mediated thrombolysis of platelet-rich clots
in vivo [90].
In rats, these ‘immediate thrombolytic effects’ of
thienopyridines were rather weak (EC
30
¼ 15–30
mgÆkg
)1
). Jean-Pierre Dupin of the Bordeaux II Uni-
versity decided to synthetize a series of thienopyrimi-

dinones under the guidance of our pharmacological
assay of their endothelium-dependent thrombolytic
effects in vivo. Assessment of their structure–activity
relationship revealed that the most active compound,
i.e. 3[(2-trifluoromethyl-phenyl)-methyl] 1,2-dihydro-
benzo[b]thieno[2,3-d]pyrimidinone-4(3H)one dissipated
platelet clots in rats in vivo at a dose of IC
30
¼
8 lgÆkg
)1
[91].
We conclude that in addition to in vivo endothelial
PGI
2
-mediated thrombolysis, thienopyrimidinones and
thienopyridines exert endothelial NO
•
-mediated coron-
ary vasodilatation in perfused guinea-pig heart [92].
Mechanisms of endotheliotropic actions of these
strongly lipophylic compounds remain unknown.
Nebivolol and carvedilol – two b
1
-adrenoceptor
antagonists – founded the ‘third generation’ of selective
b-adrenolytic drugs, which are endowed with endothe-
liotropic properties. Eleven years ago, Bowman et al.
[93] proposed that the antihypertensive effects of nebiv-
olol in man might be partially associated with endo-

thelium-dependent, NO
•
-mediated vasodilatation. In
two interesting studies Ignarro et al. [94,95] clearly
demonstrated that relaxation of vascular smooth
muscle by nebivolol is partially mediated by endothe-
lium-dependent release of NO
•
and the subsequent
accumulation of cGMP in smooth muscle [94], how-
ever, nebivolol also inhibits vascular smooth muscle
proliferation by a mechanism involving NO
•
but not
cGMP [95]. Various routes were proposed by which
the ‘third generation’ of b
1
-adrenoceptor antagonists
may release endothelial NO
•
. Certainly adrenergic and
serotoninergic receptors are not involved [96]. A fascin-
ating hypothesis has been proposed [97]. Nebivolol
and carvedilol stimulate the renal efflux of ATP,
that releases NO
•
via activation of P2Y purinoceptors
in glomerular endothelium. On top of the regular
b
1

-adrenoceptor blockade there appears NO
•
-mediated
relaxation of renal glomerular microvasculature. This
is why nebivolol and carvedilol are so efficient in
controlling arterial hypertension and improving renal
circulation.
R. J. Gryglewski Endothelium and drugs
FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2963
Perspectives
In endothelial pharmacology everything is new: (a) the
idea that vascular endothelium may be looked upon as
an organ with a secretory function; (b) considering
pulmonary endothelium as a separate endocrine organ
that supplies prostacyclin to the coronary and cerebral
circulations; (c) a complex relationship between two
endothelial mediators – NO and PGI
2
– a role for
ROS and RNS in it; (d) discovering new endothelio-
tropic mechanisms for old cardiovascular drugs like
for ACE-I, statins or nebivolol; (e) planning new
endotheliotropic chemical structures, e.g. thienopiry-
midodiones; (f) discovering new biochemical mecha-
nisms of action for drugs affecting endothelial function
like in case of nebivolol; and (g) the interaction
between basic and clinical researchers, probably one of
the most efficient in the field of medicine. Old, known
roads are safe, but the newly discovered roads are
interesting.

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