*4.2. IGF2R Modulates Vascular Remodeling and Skeletal Muscle Growth*

IGF proteins play an essential role in skeletal muscle homeostasis and vascular mechanisms involving smooth muscle cells (SMCs). The latter are driven by IGF2 in the development and migration processes occurring during vascular growth or in response to vascular damage. A study has verified the role of IGF2 in SMC migration by studying the interaction between the cellular repressor of E1A-stimulated gene (CREG) factor and M6P/IGF2-R. Specifically, when the CREG factor binds M6P/IGF2-R, it is able to inhibit the SMC proliferation process and the migration process. Further studies showed that CREG knockdown leads to an increased release of IGF2, mitigating its internalization and partly restoring the migration process of the SMCs. Accordingly, the use of an anti-human IGF2-neutralizing antibody on a CREG knockdown population promotes the inhibition (in a concentration-dependent manner) of the SMCs' migration process [50]. Despite the shortage of detailed articles, an indirect point of view of the solid connection between vascular remodeling and IGF2R is offered by Ca2<sup>+</sup> signaling. A comprehensive body of literature has already demonstrated that IGF2R triggers several intracellular signaling pathways aimed at Ca2<sup>+</sup> mobilization [41]. This cascade may occur through an increase in PKC-α phosphorylation or in a Gαq-dependent manner [51], such as for controlling hypertrophy in cardiac cells, or through PLC-induced interactions between IGF2R and G(i) protein, as in endothelial progenitor cells (EPCs) [52]. In the latter case, the upstream role of IGF2R possibly affects the migration, adhesion and invasion of EPCs in the neovascular zone, therefore raising the importance of IGF2R for vessel network formation, both in embryonic and in post-ischemic vasculo-genesis. Vascular SMCs, composing the medial layer of blood vessels, are also essential for the maintenance of post-natal vascular homeostasis, and their correct functionality is subordinated to Ca2<sup>+</sup> signaling. Intracellular calcium is likely to regulate the mechanical properties of SMCs through a tight modulation of α5β1 integrin, α-SMA and cell–ECM interactions. The Ca2<sup>+</sup> dynamic across cells may activate the elasticity and the adhesion properties of vascular SMCs, with physiologically important consequences on vascular tone and resistance, and blood flow and pressure [53].

A more complex signaling pathway underlying Ca2<sup>+</sup> entry and exit from cells [54] is tuned by ATP-dependent pumps, which counterbalance the calcium ion levels and the electrolyte homeostasis. Among these pumps, sarcoplasmic/endoplasmic reticulum (SR) Ca2+ATPase (SERCA) is the one in charge of the Ca2<sup>+</sup> homeostasis within the reticulum, with a role susceptible to the type of cells [55,56]. There are three isoforms of SERCA characterized by tissue-specific expression. Briefly, the fast twitch skeletal muscle isoform SERCA1 has been found in the heart, and, to a lesser extent, in the liver, kidney, brain and pancreas [57]. Variants of SERCA2 have been detected preferentially in cardiac, skeletal and vascular smooth muscle [58]. SERCA3 isoforms are heterogeneously expressed through tissues and, conversely to the others, are characterized by a low affinity to Ca2<sup>+</sup> [59]. Altered levels of SERCA proteins lead to aberrant calcium flux dynamics, which are responsible for the reduced contractility and dysfunction of SMCs observed in numerous diseases including cardiomyopathies, atherosclerosis, metabolic syndromes, and diabetes [60,61]. Although this evidence implies a connection between SERCA, IGF2R and calcium flux, a thorough explanation of the causal relationships is yet to be provided. Recently, we have identified a possible pathway in the context of muscular dystrophies, which are often associated with Ca2<sup>+</sup> dysfunction: the contractile function of muscle fibers is dependent on the expression of many proteins involved in the calcium cycle between the cytosol and SR. Ca2<sup>+</sup> signaling includes the ryanodine receptor, which is the SR Ca2<sup>+</sup> release channel; the troponin protein complex, which leads to contraction; the extracellular Ca2<sup>+</sup> reuptake pump, which mediates the flux of Ca2<sup>+</sup> into the SR by a mechanism called store-operated Ca2<sup>+</sup> entry (SOCE); and calsequestrin, the Ca2<sup>+</sup>

storage protein in the SR. In addition, several Ca2<sup>+</sup>-binding proteins are present in muscle tissue such as calmodulin, annexins, myosin, calcineurin and calpain [62]. In our study, we found that IGF2R expression is increased in dystrophic muscles, and IGF2R and the store-operated Ca2<sup>+</sup> channel CD20 share a common hydrophobic binding motif stabilizing their association. We verified that the intravenous administration of an anti-IGF2R-neutralizing antibody facilitates IGF2/IGF1R interactions, while the occurrence of CD20 phosphorylation activation promotes the entrance of Ca2<sup>+</sup> ions into the sarcoplasm [63]. Based on this evidence, we proposed a signaling pathway to explain the activation of SERCA and the Ca2<sup>+</sup> flux through cells. Among the pathway proteins, STIM1 and ORAI1 are engaged in SOCE regulation and activation. STIM1 acts as a sensor of Ca2<sup>+</sup> concentration in cellular stores and undergoes a horizontal movement in the SR membrane when the ER is calcium-depleted. Due to this shifting, STIM1 interacts with the membrane channel protein ORAI1 and causes calcium to enter the cell. After the replenishment of calcium stores, the ORAI1/STIM1 interaction dissolves and the Ca2<sup>+</sup> influx stops. CD20 phosphorylation decreases the interaction between CD20 and ORAI1 in store-depleted myoblasts, largely in anti-IGF2R-treated myoblasts.

In dystrophic muscle, SERCA activity is reduced [64,65] leading to higher cytoplasmic levels of calcium ions. After anti-IGF2R treatment, calcium uptake is activated by CaMKII-dependent regulation of SERCA1: the blockade of IGF2R allows the activation of calcineurin, which dephosphorylates the nuclear factor of activated T cells (NFAT), which, consequently, shuttles into the nucleus, promoting activation of the genes involved in myogenic differentiation. Moreover, the binding of anti-IGF2R to domain 11 of IGF2R activates IGF2R/Gαi2 interactions, preventing the interplay with IGF2. This event increases the bioavailability of IGF2 for IGF1R and promotes the activation of PI3-K/AKT/mTOR signaling involved in expression of myogenic genes. Our results demonstrated that the blockade of IGF2R in mdx muscles rescued the murine dystrophic phenotype and increased force production. The in vivo experiments also revealed a marked vascular remodeling consisting of structural modifications resulting in higher linearization and wrapping of muscle fibers. It was conceivable that EPCs and pericyte cells accounted for the amelioration of the microvasculature and the blood supply. Adhesion and migration of EPCs could be affected directly by the IGF2R blockade, while pericytes cells, which present a contractile activity, could sense the calcium uptake activation [66]. Initially, pericytes had been discovered as mural cells able to provide capillary stability by interacting with endothelial cells. This classification was exceeded by anatomical and morphological evidence [67] demonstrating that pericytes could not only have a contractile activity, but they could regulate blood flow, capillary diameters and vascular tone [66,68,69]. In turn, pericytes' behavior in microcirculation can be regulated by upstream and downstream signaling, depending on the tissues and cell types. For instance, in healthy conditions, pericytes' coverage of the retina is essential for maintaining the contact with the endothelium and the integrity of the vascular barrier. Diabetic mice exhibit altered retinal vasal permeability caused by high levels of macrophage-secreted cathepsin D (CD). CD has been shown to disrupt the pericyte–endothelial junction either by increasing Rho/ROCK-dependent cell contractility [70] or by binding to IGF2R via 2M6P binding sites and changing PKC-α–CaMKII signaling [71].

Additional demonstrations of the association of IGF2R with insulin resistance and glucose homeostasis are based on the discovery of IGF2R genetic variants and soluble circulating IGF2R [72] in both type 1 (T1DM) and type 2 (T2DM) diabetes mellitus [73,74]. In T2DM, hyperglycemia seems to severely affect the islet capillary pericytes in terms of reduced numbers, improper islet coverage, altered calcium flux sensitivity, and relaxation phenotype shift [75]. As a response, diabetic capillaries dilate, blood pressure increases, and islets lose the ability to adapt and control their blood flow. Finally, high glucose concentrations and streptozotocin-induced diabetic conditions lead to abnormal activation of the IGF2R pathway and a downstream signaling for the expression of proteins related to hypertrophy in cardiac tissues, and to apoptosis in cardiomyocytes [76].

Taken together, this evidence suggests the importance of IGF2R in the pathogenesis or treatment of disorders related to energy metabolism, vascular remodeling and muscle homeostasis. However, unraveling the signaling of the whole process within different tissues requires further and extensive investigation.

#### *4.3. IGF2R Is Involved in Carcinogenesis*

As described above, the proteins that constitute the IGF system—IGF1/IGF2, the surface receptors, and the IGF-binding proteins—regulate a plethora of functions related to growth and development operating through AKT1, mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) [77]. Consequently, dysfunction in this complex system is often associated with cancer. The upregulation of IGF2 was described in colorectal cancers (CRC) due to epigenetic mechanisms, and it was associated with poor survival [78]. In particular, IGF2 was dramatically overexpressed in tumorigenic clones related to IGF1, leading to constitutive activation of IGF1R and AKT [79] and to malignant modulation of apoptosis and stemness [80]. In addition, the loss of IGF2 imprinting was associated with esophageal adenocarcinoma [81], while the hypomethylation of one of the IGF2 promoters caused dysfunction in the transcriptional regulator Kruppel-like factor 4 (KLF4) in humane prostate cancer [82]. Genetic mutations in IGF2R can modify the bioavailability of IGF2 so that cancer cells can dramatically proliferate. Different studies demonstrated that IGF2R expression could be involved in the development of hepatocarcinoma, breast and ovarian human cancers by encoding for a tumor suppressor gene [83]. In particular, the loss of heterozygosity (LOH) at the M6P/IGF2R gene locus on 6q26–27 chromosome seemed to be associated with the invasiveness of breast cancers, while M6P/IGF2R point mutations were identified in hepatoma, gastrointestinal (mainly associated with microsatellite instability) and prostate tumors [84]. This condition likely led to uncontrolled IGF2 upregulation that enhanced cancer growth and survival by binding to IGF1R. In particular, the work of Delaine et al. [85] showed a new hydrophobic patch on the domain 11 of the IGF2R that is fundamental for the high binding affinity of IGF2/IGF2R. The first direct demonstration of IGF2R in tumor growth came from the work of Chen et al., in which they described how the downregulation of M6P/IGF2R expression in adenocarcinoma cells led to increased cell proliferation and decreased susceptibility to apoptosis, according to the bioavailability of TNFα and activated TGF-β. In addition, this condition was probably hampered by the action of IGF2 and cathepsins B and D [86]. Interestingly, ligands other than IGF2 can bind to IGF2R: among them are urokinase-type plasminogen activator receptor (uPAR) and retinoic acid, whose activity allows the internalization of IGF2 [7]. All the IGF2R-dependent pathways are summarized in Figure 1.

**Figure 1.** A schematic model of the IGF2R-dependent mechanisms leading to cardiac and skeletal muscle impairment, and cell sources of IGF2R expression in dystrophic muscle.

#### **5. Conclusions**

Fetal growth and post-natal growth are closely regulated by the insulin-like growth factor axis: alterations in the IGF signaling pathways could cause severe dysfunction in somatic growth and development and be responsible for tumor proliferation. We have recently demonstrated in mdx mice that intravenous administration of an anti-IGF2R neutralizing antibody significantly upregulated muscle regeneration and decreased fibrosis, leading to the rescue of the pathological phenotype. The inhibition of IGF2R resulted in an increase in intracellular Ca2<sup>+</sup> in myoblasts and increased SERCA1 activity, possibly operating through CD20. This condition allowed NFAT dephosphorylation and its translocation into the nucleus. The dystrophic phenotype rescue activated in vivo by the anti-IGF2R antibody was further corroborated by higher numbers of structurally more linear microvessels enveloping myofibers. It is likely that anti-IGF2R acts on pericyte function, determining normalization of the vascular wall and consequent amelioration of the oxygenation of the dystrophic muscle. As contractile cells, pericytes can regulate their tone and contraction depending on the intracellular calcium concentration and consequently tune the capillary diameter and blood flow [69].

This is an exciting time in our understanding of muscular dystrophies. Increasing knowledge of IGF2R's role in muscle disease is starting to suggest new therapeutic approaches. A challenge for the future will be to understand how IGF2R interacts with other components of the muscle system to influence muscular dystrophy progression. Similarly, genetic and epigenetic changes affecting *IGF2R* need to be considered. Therefore, targeting IGF2R may be a potential therapeutic strategy for muscular dystrophies.

**Author Contributions:** A.F., Y.T. and C.V. wrote the paper. P.B. and L.T. prepared the original draft of the manuscript. All the authors stated were involved in the critical revision of the manuscript and approved the final version of the article, including the authorship list. The corresponding authors had final responsibility for the decision to submit for publication. All authors have read and agreed to the published version of the manuscript. **Funding:** This study was supported by the Associazione Centro Dino Ferrari.

**Acknowledgments:** Funders of the study had no role in manuscript design.

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