DISCUSSION AND CONCLUSIONS
Our experimental study shows, for the first time, that acquired CCN2
deficiency potentiates Ang II-induced deleterious effects in the aorta,
increasing the risk of life-threatening TAAAs aneurysm formation and
dissection. In addition, spironolactone preventive treatment increases
the survival rate and decreases aortic aneurysm formation in CCN2-KO
mice after Ang II administration, suggesting a main role of the
mineralocorticoid receptor signaling in this pathological process. The
observed beneficial effects of spironolactone in this new
aneurysm-formation experimental model suggest the mineralocorticoid
receptor antagonist treatment as a potential therapeutic option in
aneurysms-related human pathologies.
CCN2 plays a key role in the regulation of ECM proteins, as observed in
embryonic vascular development, regulating elastin and different types
of collagen, and stabilizing mature vessels (Chaqour, 2013). Many
preclinical studies have demonstrated that CCN2 is involved in fibrotic
disorders (Luo et al., 2008; Ponticos et al., 2009; Phanish et al.,
2010; Hao et al., 2014; Huang et al., 2016). However, CCN2 modulation
can exert opposite effects depending on the pathological conditions, as
described in various cardiovascular pathologies (Gravning et al., 2013;
Moe et al., 2016). In a previous in vitro study, we found that
Ang II infusion produced collagen accumulation and CCN2 overexpression
in rat aorta, and CCN2 blockade diminished Ang II-induced ECM
overproduction in culture vascular smooth muscle cells (Rupérez et al.,
2003). At human level, CCN2 upregulation has also been described in
aortic aneurysms (Branchetti et al., 2013), and it was associated with
collagen deposition in human atherosclerotic lesions and in thoracic
aortic dissection (Ponticos, 2013; Meng et al., 2014). Our present
findings showing exacerbated aortic aneurysm formation and fatal
dissection caused by Ang II in CCN2 deficient mice point out a potential
role of CCN2 in the regulation of an adaptive and protective response
that maintains aortic wall integrity. The histological evaluations of
CCN2-KO mice revealed aortic wall alterations, including elastic layer
disruption, which were more severe after Ang II infusion. Elastic fibers
are major aortic ECM components, and their degradation or loss alter the
mechanical behavior of the aortic wall (Yanagisawa and Wagenseil, 2020).
Enzymes involved in ECM degradation, such as MMPs and collagenases, have
special relevance in aneurysm formation and rupture (Matthew Longo et
al., 2002; Ju et al., 2014; Thirunavukkarasu et al., 2016). Here, we
described that acquired CCN2 deficiency increased MMP2 and MMP9
activity, and upregulated MMP8 synthesis. The elevated MMPs activity was
located between the elastic layers, coincidentally with elastin loss.
Previous studies demonstrated that Ang II increased aortic MMPs activity
and elastic layer degradation (Forrester et al., 2018), as we have also
found in CCN2-deleted mice. These findings support that MMPs regulation
is also an important mechanism in CCN2-mediated aortic wall
destabilization and aneurysm formation.
Several observational studies reported dysregulated CCN2 expression in
both non-syndromic (Branchetti et al., 2013) and syndromic human
thoracic aortic aneurysms, as observed in several heritable connective
tissue disorders such as Marfan, Loeys-Dietz, Ehlers–Danlos,
aneurysms-osteoarthritis, and the arterial tortuosity syndrome (Zoppi et
al., 2018). These pathologies are characterized by altered connective
tissue, resulting in perturbed assembly of ECM, maintenance and
homeostasis in various organ systems, including blood vessels, heart,
bones, eyes, skin or lungs (Zoppi et al., 2018). In some of these
syndromes, aneurysms have been linked to TGFβ pathway deregulation.
Marfan syndrome is caused by mutations in the gene that encodes
fibrillin-1, which participates in the maintenance of the large latent
complex of TGFβ1 in its inactive state. The Loeys-Dietz Syndrome is
associated with heterozygous mutations in the genes encoding TGFβ
receptors (TGFBR) 1 and 2, and other components of the pathway (TGFB2,
TGFB3, SMAD3, and SMAD4) (Loeys et al., 2005). Mutations in those genes
are expected to disrupt TGF-β signaling. Paradoxically, increased TGFβ
signaling was suggested to cause aortic aneurysm development in those
syndromes; however, approaches disrupting TGFβ in experimental aneurysm
models were not protective (Chen et al., 2016; Lareyre et al., 2017;
Mallat et al., 2017). In fact, TGF-β blockade, as observed here for CCN2
deletion, exacerbates aneurysm formation and dissection in several
experimental models(Wang et al., 2010; Li et al., 2014; Mallat et al.,
2017), although full mechanisms have not been completely unraveled.
Moreover, although some authors suggested that angiotensin II receptor 1
blockade should be considered in patients with thoracic aortic aneurysms
based on its ability to inhibit TGFβ production (Habashi et al., 2006),
clinical results are controversial, as losartan did not provide any
benefit in Marfan patients but irbesartan was associated with a reduced
rate of aortic dilatation in children and young adults with this
syndrome. (Lacro et al., 2014; Milleron et al., 2015; Mullen et al.,
2019). Interestingly, another study revealed that human abdominal aortic
aneurysm was associated with down-regulation of transcripts encoded by a
16-Mbp segment between cytogenetic bands q22.1 and q23.2 of chromosome
6, where only CCN2 was found differentially expressed (Biros et al.,
2014). Therefore, authors suggested that CCN2 could be directly related
to aneurysm generation in these patients. The present findings show that
CCN2 deletion shares similarities to TGFβ pathway disruption in aortic
aneurysm development, providing a novel framework to explore the
pathogenesis of aneurysms and aortic rupture. Thus, our results suggest
that the safety of CCN2 blockade should be studied in depth prior to
considering it as a therapeutic option in patients predisposed to or
presenting aortic aneurysm.
Hypertension is a well-known predisposing risk factor for human thoracic
aortic disease (Lemaire and Russell, 2011), although mechanisms involved
are not well elucidated. Hypertension resulting from activation of the
renin-angiotensin system has been associated with both abdominal and
thoracic aortic aneurysms in experimental mice models under certain
circumstances, in particular in response to Ang II administration in
Apoliprotein E deficient mice (Trachet et al., 2015). Here, we show that
the resistance of young C57Bl/6 mice to Ang II-induced aortic aneurysm
is lost in a context of CCN2 deficiency. Importantly, CCN2 deletion did
not increase Ang II-induced blood pressure elevation as compared to WT
mice, suggesting that the deleterious effects of CCN2 absence are
independent on blood pressure regulation. On the contrary, systolic
blood pressure decreased in CCN2-KO mice compared to WT mice. These
results agree with observations in genetically modified mice of another
ECM components such as the extracellular metalloproteinase ADAMTS1.
Thus, studies of partial deletion or silencing of this gene, showed that
ADAMTS1 reduction was associated to blood pressure decrease and aneurysm
formation in response to Ang-II infusion (Oller et al., 2017), as we
have observed here in CCN2 deleted mice.
The role of some other CCN family proteins in the pathophysiology of
aneurysm generation have been recently described. CCN4 deletion
suppressed aneurysm severity in ApoE knockout mice infused with Ang II
(Williams et al., 2021). Though CCN3, in the same way as we observe
here, seemed to be protective in two different experimental models of
aneurysm, induced by elastase and Ang II administration. All these data
highlight the functional diversity of these factors. Our results suggest
that CCN2 is an important matricellular ECM protein necessary for
maintaining the adult vascular wall architecture in health and disease,
supporting its key role as a regulator of vascular ECM matrix
homeostasis.
One of the most enriched biological process terms in the absence of CCN2
was the aldosterone biosynthetic process, suggesting that aldosterone
could participate in the observed aneurysms pathogenesis. Several
clinical cases revealed aortic dissection in primary aldosteronism
patients (Ahmed et al., 2007). Some studies found an association between
mineralocorticoid receptor blockers and slowed aortic aneurysm
progression (Thompson et al., 2010; Kurobe et al., 2013). In mice fed a
high salt diet, treatment with a mineralocorticoid receptor agonist
caused aortic aneurysm formation and rupture (Liu et al., 2013). Here,
we demonstrate that mineralocorticoid receptor inhibition by
spironolactone reduced aneurysm generation and death induced by Ang II
infusion in CCN2-KO mice, without preventing blood pressure elevation.
Accordingly, previous studies have described that another
mineralocorticoid receptor antagonist, eplerenone, significantly reduced
aneurysm development induced by combination of Ang II and
β-aminopropionitrile in mice, without affecting blood pressure (Kurobe
et al., 2013). Regarding the CCN2-TGF-β connection, there is an
interesting relation between aldosterone, the TGF-β pathway and CCN2.
Aldosterone increased TGF-β synthesis (Juknevicius et al., 2004;
Ruiz-Ortega et al., 2007b) and induced CCN2 in mice cardiomyocytesin vivo and in experimental diabetic nephropathy (Han et al.,
2006; Messaoudi et al., 2013). By contrast, TGF-β is a potent suppressor
of multiple enzymatic steps of steroidogenesis upstream of aldosterone
synthesis, with targets including Cyp11a1 , Cyp11b1,
Cyp11b2, Cyp21a1, NR5A1, FDX1, FDXR, StAR or Hsd3b1 (Matsuki et
al., 2015). Our findings showed that several of these genes were
upregulated in the aorta of CCN2-KO mice, suggesting a similar role of
CCN2 in regulating the aldosterone pathway as previously described for
TGF-β. Furthermore, our data postulate the involvement of the
aldosterone pathway in aortic aneurysm generation and severity, and
support future studies to investigate the potential use of
mineralocorticoid receptor antagonists as therapeutic targets in the
aneurysm context. OK
In conclusion, we clearly demonstrate, for the first time to our
knowledge, that the absence of CCN2 expression predisposes mice to a
pathological response to Ang II that favors aneurysm generation and
rupture, which is partially mediated by a deregulation of the
aldosterone synthesis pathway. Furthermore, considering the
well-established connection between TGF-β signaling and CCN2, our
current study further supports the importance of the TGF-β pathway in
vascular integrity protection against the development and progression of
aortic aneurysm and suggests a potential role of the aldosterone pathway
in this process. These results open new approaches to study
cardiovascular diseases related to connective tissue disorders,
especially those associated with vascular abnormalities and aneurysm
formation, and sustain the use of mineralocorticoid receptor antagonists
as a potential therapeutic approach in their pathogenesis.