**2. Summary and Outlook**

Dissecting out novel pathways of vascular damage dependent on a gene variant is an intriguing issue from both a scientific and a clinical point of view. In the case of T2238C/ANP, we demonstrated that the extended (30-amino acid) gene product acts through the activation of a specific receptor (the NPR-C receptor) to induce multiple deleterious mechanisms. In addition, the ANP variant targets several cell types belonging to both the vascular wall and the bloodstream and, by doing so, it increases the cardiovascular risk (Figure 1).

**Figure 1.** Schematic representation of the molecular signaling pathways underlying the effects of CC2238/αANP in endothelial cells, smooth muscle cells, and platelets. Abbreviations: AC, adenylate cyclase; Akt, protein kinase B; APO-E, apolipoprotein E; ATP, adenosine triphosphate; BCL-2, B-cell lymphoma 2; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CREB, cAMP response element-binding protein; DNAJA4, DnaJ (Hsp40) homolog; Egr-1, early growth response protein-1; eNOS, endothelial nitric oxide synthase; GC, guanylate cyclase; GTP, guanosine triphosphate; Gαi, inhibitory G protein; miR-21, microRNA-21; miR-199, microRNA-199; NADPH OX, nicotinamide adenine dinucleotide phosphate-oxidase; NO, nitric oxide; NOX2, NADPH oxidase 2; NPR-A/B, natriuretic peptide type A/B receptors; NPR-C, natriuretic peptide type C receptor; P-AKT, phosphoprotein kinase B; PDCD4, programmed cell death protein 4; PKA, protein kinase A; PKG, protein kinase G; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species; VSMCs, vascular smooth muscle cells; αANP, carboxy-terminal atrial natriuretic peptide, wild-type (T2238) and mutant (C2238). Arrows indicate the positive stimulation within each pathway. The dotted arrow indicates lower affinity binding of C2238 for NPRA/B.

Interestingly, the findings describe for the first time the cAMP-PKA axis as the one driving all other signaling events stemming from CC2238/αANP, such as the CREB-miR21and the Egr-1-miR199a-ApoE pathways in smooth muscle cells, NADPH increase, and particularly Nox2 increase in platelets (Figure 1). In vivo, we obtained evidence of reduced endothelial-dependent vasorelaxation in apparently healthy subjects, of increased platelet activation and aggregation in a cohort of patients with atrial fibrillation, and of higher residual platelet aggregation in ischemic heart disease diabetic patients (following administration of dual antiplatelet therapy) if they were carrier of the C2238/αANP variant [55]. A previous pharmacogenomic study had already revealed the greater efficacy of diuretic therapy in hypertensive carriers of the C2238/ANP variant allele [28].

The findings provide original evidence in the field of the pathogenesis of vascular damage. We believe that the complex, multifaceted mechanisms explaining the negative impact of a molecular variant of ANP give even more support to the beneficial role played by the common form of ANP within the cardiovascular system. In fact, we can better support the view that the common form of ANP, acting through NPR-A activation and the consequent cGMP release, is a peptide promoting vascular health, angiogenesis, vascular smooth muscle cells relaxation, regular platelets activation and aggregation. On the other hand, the change in the peptide sequence leads to higher binding affinity for NPR-C and introduces several differences in the functions of the peptide, making C2238/ANP a "toxic" substance for the vascular wall and for the cardiovascular system generally [57].

A few final considerations are needed in regard to what we have learned about the T2238C/ANP variant. First of all, discovering a functional biological effect of a gene variant is not common. Furthermore, the translation of in vitro effects to in vivo condition is even less likely. Even when this achievement is obtained, the expected effect in vivo is usually small in the context of complex multifactorial diseases.

The case of CC2238/αANP is uncommon. In fact, apart from dissecting out its specific biological effects, the molecular studies allowed the discovery of a novel role of a known receptor (NPR-C) and of novel signaling pathways leading to vascular damage. More importantly, the deleterious impact of the alternative peptide can be detected in vivo, which is uncommon and likely to be of significant relevance to the physiological effects of ANP.

Therefore, the current mechanistic knowledge on C2238/αANP underscores a novel cardiovascular genetic risk factor that needs to be taken into account in daily clinical practice. Of note, the cost of the determination of the presence of the T2238C allele is affordable.

Further studies should assess whether T2238C allele characterization may add some value on top of the conventional risk factors and may have clinical and therapeutic relevance.

Moreover, some of the molecules outlined in our studies could be targeted by novel therapeutic approaches to reduce the cardiovascular risk in carriers of the C2238/ANP allele.

**Acknowledgments:** This work was supported by a grant from the Italian Ministry of Health and from the 5grant.

**Author Contributions:** Speranza Rubattu prepared the article, Sebastiano Sciarretta, Simona Marchitti, Franca Bianchi, Maurizio Forte and Massimo Volpe revised it. Maurizio Forte prepared the figure.

**Conflicts of Interest:** The authors declare no conflicts of interest.
