IGF-I ESTIMULA LA LIBERACIÓN DE APOE POR ASTROCITOS

1.8.1 Overview

The relatively high expression of CNP in endothelial cells and the presence of its receptor on the underlying smooth muscle cells suggest that CNP plays a role in vascular homeostasis (Stingo et al., 1992). Due to the low plasma levels of CNP, it is thought to act in a paracrine/autocrine fashion. In healthy human subjects, the plasma concentration of CNP is approximately 1 pg/ml (Igaki et al., 1996, Hama et al., 1994) and rises in pathological conditions, especially in patients with cardiovascular disorders (septic shock, 13 pg/ml (Hama et al., 1994); renal failure, 3 pg/ml (Igaki et al., 1996); CHF, 8 pg/ml (Del Ry et al., 2011a)), diabetes (9 pg/ml (Del Ry et al., 2011a)) and cirrhosis (5 pg/ml (Del Ry et al., 2011a)). In conditions of oxidative stress, such as atherosclerosis, NO bioavailability is reduced whilst CNP production is increased (Chun et al., 2000). These observations suggest CNP may play a protective, compensatory role to NO in maintaining endothelial function.

1.8.2 CNP in the regulation of vascular tone

CNP is widely reported as a potent vasodilator in various vascular beds of different species including human (Wiley and Davenport, 2001), pig (Barber et al., 1998), rat (Chauhan et al., 2003) and mouse (Madhani et al., 2003). In isolated porcine coronary arteries CNP evokes vasorelaxation by hyperpolarisation (Barton et al., 1998). Subsequently, Chauhan et al.

(2003) showed that CNP and EDHF exhibit equivalent hyperpolarisation and relaxation responses in rat mesenteric artery that were blocked by well-characterised EDHF inhibitors.

This provided the first evidence that CNP acts as an EDHF and regulates local vascular tone in parallel with NO and PGI2. Correspondingly, mice lacking endothelium-derived CNP have reduced vasorelaxant responses to ACh compared to WT in the presence or absence of NO and PGI2 blockade (Moyes et al., 2014). With the use of the selective NPR-C antagonist, M372049, it has been revealed that CNP acts on NPR-C leading to Gi-dependent activation of Ba²⁺-sensitive GIRK channels and Na⁺/K⁺-ATPase in the VSMC, resulting in hyperpolarisation and vasorelaxation (Villar et al., 2007) (Figure 8). This mechanism represents a major component of EDHF-induced smooth muscle relaxation. However, activation of proteinase-activated receptor 2 (PAR2) in the endothelium also mediates EDHF responses and this is not altered in mice deficient in the NPR-C gene (McGuire et al., 2004).

Furthermore, vasodilator responses to CNP in capillaries are preserved in endothelial-restricted NPR-B KO mice but abolished in pericyte NPR-B KO (Spiranec et al., 2018). These

observations indicate CNP signalling is important in regulating different vascular cell types across various vascular beds.

1.8.3 CNP in the regulation of blood pressure

The ability of CNP to regulate vascular tone intimates that this peptide contributes to BP regulation. In a genetic association study, it has shown that a single-nucleotide polymorphism in the CNP gene associates with hypertension, particular in younger adults (Ono et al., 2002). GWAS have also shown that mutations in NPR-C and furin associate with higher BP (Ehret et al., 2011). This is consistent with animal studies that have revealed disruption of CNP production leads to a hypertensive phenotype in an allele dependent manner, particularly in female animals (Moyes et al., 2014).

1.8.4 Interaction between CNP and renin-aldosterone-angiotensin system

There is evidence that CNP contributes to the regulation of BP by interfering with the RAAS signalling in a manner akin to ANP. Ang II stimulates the secretion of ET-1, a process that is inhibited by CNP with a greater potency than ANP or BNP (Kohno et al., 1992). In human forearm blood flow studies, CNP inhibits Ang I-induced vasoconstriction but not Ang II, intimating that CNP inhibits ACE activity (Davidson et al., 1996). On the other hand, ramipril, an ACEi, increases CNP mRNA expression in the renal cortex (Walther et al., 2001), suggesting the use of ACEi not only inhibits the generation of Ang II but also increases CNP expression that could be cardioprotective. However, the overall diuretic and natriuretic effect of CNP is modest compared to ANP and BNP (Kalra et al., 2001).

1.8.5 CNP in vascular cell proliferation and remodeling

Atherosclerosis is a huge contributor to cardiovascular disorders including CHD, stroke and MI (Hansson, 2005). To promote endothelial regeneration whilst inhibiting VSMC proliferation is essential to prevent atherosclerosis, restenosis and maintain a healthy vasculature (Kipshidze et al., 2004, Losordo et al., 2003). Many studies have shown that CNP is a strong anti-proliferative agent and reduces intimal thickening via the NPR-B/cGMP cascade (Furuya et al., 1993, Doi et al., 2001, Ohno et al., 2002). Similar results were reported in different vascular beds and species, including rat and rabbit carotid artery (Furuya et al., 1993, Gaspari et al., 2000, Shinomiya et al., 1994), porcine coronary artery (Morishige et al., 2000) and porcine femoral artery (Pelisek et al., 2006). However, a study by Cahill et al. (1994) showed that CNP suppresses aortic smooth muscle cell proliferation via a cGMP-independent pathway involving NPR-C. This result is supported by recent

studies from the Hobbs’ laboratory, using antagonists and NPR-C KO mice, demonstrating that NPR-C dependent ERK1/2 phosphorylation is responsible for the dual effect of CNP to inhibit VSMC proliferation but concomitantly augment endothelial cell growth (Khambata et al., 2011). These dual roles of CNP in vascular cell proliferation might represent an attractive therapeutic avenue in atherosclerosis and might provide an alternative agent to the existing drug eluting stents for patients undergoing PCI.

1.8.6 CNP in vascular inflammation and atherosclerosis

One of the early steps of atherogenesis is the rolling and adhesion of leukocytes to the endothelial surface through interaction of various cell adhesion molecules that are expressed on both leukocytes and endothelial cells (Libby, 2002). CNP has been shown to supress leukocyte rolling induced by acute inflammation or in a high basal leukocyte activation mouse model (i.e. eNOS KO mice). Genetically modified mice with endothelial CNP deletion exhibit increased basal leukocyte rolling, and apolipoprotein E (ApoE)/endothelial CNP double KO mice are more prone to the development of atherosclerotic plaque in the aorta compared with WT/ApoE KO littermate controls (Moyes et al., 2014). These findings provide strong evidence that CNP has anti-atherosclerotic properties.

Furthermore, CNP is expressed strongly in coronary atherosclerosis lesions, particularly in the endothelium, VSMCs and macrophages (Casco et al., 2002). Interestingly, the level of CNP expression correlates with the severity of human atherosclerotic lesions. Naruko et al.

(1996) showed that CNP expression is present in the endothelial cells of non-lesional human coronary arterial segments, but is decreased in lesional segments (Naruko et al., 1996). Conversely, CNP levels are increased in VSMCs and macrophages of the lesion area but absent from healthy segments (Naruko et al., 1996, Casco et al., 2002). In addition, NPR-A is absent in the lesions but both NPR-B and NPR-C are expressed (Casco et al., 2002).

Taken together, this evidence indicates an autocrine/paracrine action of CNP in modulating the progression of atherosclerotic disease via its anti-proliferative and anti-migratory action on VSMCs and macrophages, and maintenance of vasculature integrity.

EDHF/CNP-mediated vascular smooth muscle hyperpolarisation

Figure 8. EDHF/CNP-mediated vascular smooth muscle hyperpolarisation.

EDHF/CNP is released in the endothelial cell upon activation of calcium-dependent potassium channel, SKCa.

The release of CNP results in the activation of NPR-C on the vascular smooth muscle cell, which causes activation of Na⁺/K⁺ ATPase and G protein-gated inwardly rectifying potassium (GIRK) channels via Gi-coupling, leading to hyperpolarisation and vasorelaxation. SKCa, calcium activated small-conductance potassium channel;

BKCa, calcium activated big-conductance potassium channel; CTx, charybdotoxin; PTx, pertussis toxin.