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.