3. ACE2 and Bradykinin
ACE2 is a membrane-associated aminopeptidase and belongs to the
angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases
and has high homology to human angiotensin 1 converting enzyme (ACE1)
(Tipnis et al . 2000). A region of the extracellular portion of
ACE2 that includes the first α-helix and lysine 353 and proximal
residues of the N terminus of β-sheet 5 interacts with high affinity to
the receptor binding domain of the SARS-CoV S glycoprotein (Li et
al . 2005b). The secreted ACE2 catalyzes the cleavage of angiotensin I
into angiotensin 1-9, and angiotensin II into the vasodilator
angiotensin 1-7, explaining the negative regulation activity exerted on
angiotensin II-induced increase of blood pressure (Patel et al .
2016). Beyond its role in the cardiovascular system, it plays a role in
the regulation of renal function and fertility (Koitka et al .
2008; Pan et al . 2013). Of recent outbreak, it was demonstrated
that ACE2 plays as a functional receptor for the S glycoprotein of the
human coronavirus SARS-CoV2 (COVID-19 virus) (Imai et al . 2005;
Zhou et al . 2020b). Once the virus binds with its glycoprotein
moiety of the extracellular S protein to ACE2, it is endocytosed into
the host cell allowing its reproduction, leading to ARDS- and
SARS-induced pulmonary edema. The ligation of ACE2 by the virus provides
ACE2 blockade which systemically translates into higher hypertensive
activity of Angiotensin II, which is not catabolized into angiotensin
1-7 or angiotensin 1-9, promoting not only the well-known hypertensive
and hypertrophic activity on the cardiovascular system, but also the
leakage of pulmonary blood vessels, as demonstrated in an in vivomodel (Imai et al . 2005) (Figure 1). This likely leads to what we
are actually assisting in terms of high blood pressure in COVID-19
patients and pulmonary edema up to angioedema, which underlies the fact
that ACE2 cleaves a single-terminal residue of several bioactive
peptides, such as neurotensin, dynorphin A (1-13), apelin-13, and
des-Arg9bradykinin (DABK) (Vickers et al . 2002;
Donoghue et al . 2003). Herein, once ACE2 is blocked by SARS-CoV2,
besides the perturbation of the pulmonary renin-angiotensin system (RAS)
(Imai et al . 2005; Kuba et al . 2005), increasing
inflammation and vascular permeability occur, due to the activity of
DABK that binding to bradykinin receptor B1 (BKB1R) can lead to acute
lung inflammation (Sodhi et al . 2018) (Figure 1). In support, in
a mouse model of endotoxin inhalation, the absence of ACE2 led to the
activation of the DABK/BKB1R axis, release of pro-inflammatory
chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage
inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (CXCL1), also
known as keratinocyte-derived chemokine (KC), and TNF-α from airway
epithelia, increasing neutrophilia and inflammation with an ensuing lung
injury. To date, the same authors showed that endotoxin administration
in mice induced ACE2 attenuation in the lung partly due to NF-κB
signaling, which is constantly activated during an acute inflammatory
process, especially by IL-1β, as well as IL-6, and other
pro-inflammatory cytokines, exacerbating lung inflammation/edema up to
organ dysfunction (Sodhi et al . 2018).
Therefore, if we consider the clinical outcome of COVID-19 patients, we
could speculate that the blockade or under-expression of ACE2 by
SARS-CoV2 and the ensuing pro-inflammatory mediators (i.e. IL-1β and
IL-6) contribute to the pathogenesis of lung inflammation because of an
impaired catabolism of DABK, leading to an enhanced BKB1R signaling,
resulting in what is actually called “cytokine storm” during the early
onset of COVID-19. Bradykinin receptors are B1, an induced receptor
during inflammatory conditions, and B2, a constitutive and ubiquitous
receptor (Marceau, Hess, and Bachvarov, 1998). B2 receptor mediates the
action of BK and lysyl-bradykinin (Lys-BK), the first set of bioactive
kinins formed in response to injury from kininogen precursors through
the action of plasma and tissue kallikreins; whereas B1 receptor
mediates the action of DABK and Lys-DABK, the second set of bioactive
kinins formed through the action of carboxypeptidases on BK and Lys-BK,
respectively (Couture et al . 2001). The B2 receptor is ubiquitous
and constitutively expressed, whereas the B1 receptor is expressed at
very low levels in healthy tissues but it is induced following injury by
various pro-inflammatory cytokines such as IL-1β (Marceau , Hess, and
Bachvarov, 1998). Both receptors act through Gαq to
stimulate phospholipase Cβ followed by phosphoinositide hydrolysis and
intracellular free Ca2+ mobilization, and through
Gαi to inhibit adenylate cyclase and stimulate the MAPK
pathways (Leeb-Lundberg, 2004) (Figure 2). Although little is known
about the cross-talk between B1 and B2 receptors, it is well established
that B2 signalling can mediate B1 upregulation via MAPK- and
NF-kB-dependent pathways, and that the expression of both receptors can
be induced by pro-inflammatory cytokines (i.e. TNFα and IL-1β), creating
a “catch-22 loop ” (Brechter et al . 2008). This molecular
mechanism/s translated into clinical outcomes underlie vascular
permeability and dilation, bronchoconstriction (cough) and pain
(hyperalgesia, muscular pain) with ensuing fever due to the cytokine
storm, all symptoms of COVID-19. (Figure 1).
An important issue is that BK is produced by the kallikrein-kinin system
(KKS). The KKS consists of prekallikrein in complex with high molecular
weight kininogen (HMWK) (Hooley, McEwan, and Emsley, 2007) (Figure 2).
HMWK is a multifunctional single-chain plasma glycoprotein primarily
expressed by the liver and secreted into the bloodstream. HMWK consists
of 6 different proteic domains (Shariat-Madar and Schmaier, 1999) and
binds to prekallikrein by means of a sequence in domain 6. The
detachment of the domain 4 liberates BK (Griffin and Cochrane, 1979).
Kallikreins are serine proteases responsible for the release of kinins,
vasoactive peptides that cause vascular smooth muscle relaxation and an
increase of vascular permeability (Bhoola, Figueroa, and Worthy, 1992).
It has been found that kallikrein exists in two different forms: plasma
kallikrein, which cleaves HMWK into BK, which in turn interacts with the
constitutive B2 receptor, and tissue kallikrein which processes
low-molecular-weight kininogen (LMWK) into Lys-BK. The interaction of BK
or Lys-BK onto B1 and B2 receptors will increase the activation of both
endothelial NOS (eNOS) and inducible NOS (iNOS), with an ensuing release
of nitric oxide, potent vasodilator, of prostaglandin I2 (PGI2) and
pro-inflammatory cytokines and chemokines responsible for acute
inflammation that is accompanied by vasodilation, pain, cell
proliferation and fibrosis (Kuhr et al . 2010; Tsai et al .
2015), symptoms typical of COVID-19 (Figure 1; Figure 2).
Plasma as well as tissue kallikrein are initially secreted as inactive,
but both of them are activated by serine protease activity (Bhoola,
Figueroa, and Worthy, 1992). The reciprocal activation of Factor XIIa
(Hageman Factor) and plasma prekallikrein promotes the activation of the
kallikrein, which, besides the catabolism of HMWK into BK, initiate the
intrinsic pathway of coagulation, influencing fibrinolysis (Figure 2).
At the same time, tissue pre-kallikrein cleaves low molecular weight
kininogen (LMWK) in des-Arg-kallidin and des-Arg9-BK
which interact with B1 receptors further enhancing inflammation. The
intrinsic pathway of coagulation is then correlated to the extrinsic
pathway of the coagulation in that Factor XIIa activates Factor XI,
which leads to the activation of factor IX which subsequently leads into
the common pathway by the activation of Factor X and then thrombin, with
fibrin aggregates generation, hence the need to detect D-dimer in
COVID-19 patients (Figure 2). In this context, studies looking at rat
models that express both BK receptors show, in vitro , that BK
acting through the B2 receptor on the surface of endothelial cells
promotes the expression of procoagulant and antifibrinolytic proteins,
such as tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1)
(Kimura et al . 2002). On the other hand, plasma kallikrein can
align pro-urokinase plasminogen activator (u-PA) in such close proximity
as to drive plasminogen activation into plasmin which degrades fibrin
aggregates (Selvarajan et al . 2001), effects that are widely
observed in sepsis, another co-morbidity of COVID-19. However, it has
been shown that the complex HMWK and Factor XIIa can also bind to
another of the three endothelial cell-binding sites, the 33-kDa cell
surface receptor for the first component of complement C1q (gC1qR/p33)
which has high affinity for HMWK (Ghebrehiwet et al . 2006).
Therefore, the activation of the classical pathway of the complement
together with the activation of the plasmin on the conversion of C3 into
C3a and C3b induce the activation of both lecithin and extrinsic
pathways of the complement with the ensuing activation of the humoral
immunity, exacerbating the inflammatory process (Figure 2).
These events may happen in COVID-19 patients from the early onset up to
the severe step of the pathology. To date the above pathological
conditions are typical of angioedema, cardiovascular dysfunction and
sepsis, pathologies which symptoms occur in COVID-19 patients. But it is
obvious to ask the correlation between these symptoms and the viral
infection. Why would this happen? Our hypothesis is that the infection
by SARS-CoV2 that “uses” ACE2 to enter the host, blocks the activity
of the degradation of angiotensin II, but at the same time the
degradation of BK is altered and impaired in that to trigger and enhance
all the above described clinical events.