The hormone ghrelin was first isolated from rat stomach in 1999 and was subsequently identified as the endogenous ligand for the GHS-R type 1a (21). Ghrelin is predominantly produced by P/D1-like (X/A-like cells in the mouse) cells in the oxyntic glands of the gastric mucosa, but is also expressed in a smaller amount in other tissues including the myocardium, arteries, and veins (25, 26). Ghrelin circulates in two distinct forms: acylated ghrelin (AG) and des-acylated ghrelin (Des-AG). AG possesses a novel octanoylation of serine-3, promoted by ghrelin O-acyltransferase (Figure 1). This is considered essential for facilitating the binding of the peptide to the GHS-R and eliciting its physiological functions (53). Des-AG lacks GH-secretagogue activity and does not bind to the GHS-R type 1a. The mechanisms by which Des-AG exerts its biological functions remain largely unknown, as its target receptor is yet to be identified. Emerging evidence from the literature consistently advocates the therapeutic potential of ghrelin as a novel strategy for the treatment of various metabolic and cardiovascular disorders. These include anorexia, sarcopenia, cardiomyopathy, CLI, inflammatory disorders, metabolic syndrome, epilepsy, and neurodegenerative disorders (54–60).

Ghrelin has been shown to have a pro-angiogenic potential in several studies (22, 61–64), but limited potential in others (65–67). Ghrelin’s angiogenic potential was first shown in human microvascular endothelial cells (HMVEC) at a concentration of 0.1 nM. At this concentration, it promoted migration, proliferation, and angiogenesis through phosphorylation and activation of the mitogen activated protein kinases ERK2, which regulates endothelial cell function (63). The same research group has also demonstrated that AG at 1 nM significantly reverses age-related impaired angiogenesis in HMVECs. This is mainly through the activation of the MAPK/ERK2 mitogenic signalling pathway, a central pathway for angiogenesis which promotes endothelial cell motility and survival (62). Using cardiac microvascular endothelial cells, AG at concentrations of 10 nM significantly stimulated proliferation, migration, and angiogenesis in vitro. This was through the GHS-R1a type 1a-mediated kinase MEK and extracellular signal-regulated kinase ERK, in addition to the most commonly studied pro-survival phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway, which regulates numerous cellular processes, including cell cycle, angiogenesis, and apoptosis (68). Taken together, these findings indicate exogenous ghrelin administration can activate several independent angiogenesis signalling pathways (61).

In a rat model of chronic myocardial ischemia, following permanent occlusion of the left anterior descending artery, AG (100 µg/kg, twice daily for 4 weeks) increased VEGF expression, inhibited apoptosis, and increased angiogenesis in the myocardial infarct and peri-infarct zones (64). In a murine model of CLI, AG (150 µg/kg, daily for 2 weeks) promoted angiogenesis through the up-regulation of pro-angiogenic microRNAs (miRs)-126 and-132, while preventing the activation of antiangiogenic miRs-92a and -206 (22).

Interestingly, Des-AG (100 µg/kg daily for 2 weeks) also exerted a protective vascular effect in a murine model of CLI, evident from a comparatively similar blood vessel density in the ischemic versus contralateral (non-ischemic) limbs (69). However, Des-AG did not change large vessel perfusion or induce neovascularization, possibly due to its up-regulation of antiangiogenic miRs-221 and -222 (70). This suggests Des-AG may limit adverse changes in the vasculature in response to ischemia, rather than initiate angiogenesis (69, 71).

Although the majority of studies advocate the angiogenic potential of AG, conflicting reports from one research team propose that AG may have antiangiogenic properties. This is based on the observation that AG (10 nM) impeded the in vitro activation of angiogenesis induced by fibroblast growth factor-2 in human umbilical vein endothelial cells (HUVEC), and in rat neuromicrovascular endothelial cells, via inhibition of tyrosine kinase and MAPK pathways (65, 67).

The discrepancy between studies, especially in vitro studies, concerning the angiogenic properties of ghrelin is unquestionably confounded by experimental factors. These include differing cell types, culture conditions, different concentrations, and differing methodological approaches. However, it is clear from in vivo animal models of MI and CLI that ghrelin exerts beneficial effects through vascular protection and neovascularization.

Impaired perfusion of the lower limbs results in significant atrophy of the calf muscle and increased fat composition, causing severe functional impairment (72). Circulating levels of ghrelin are reduced in diseases of muscle wasting (650 vs. 899 pg/mL) (73). Yet, the therapeutic administration of exogenous ghrelin in a murine model of muscular atrophy induces Akt^S473 phosphorylation- a direct anti-atrophic signalling pathway in skeletal muscle, blocking skeletal muscle atrophy (74). In murine models of CLI, Des-AG has been shown to induce skeletal muscle regeneration through increased superoxide-dismutase-2 (SOD-2) induced expressions of miRs-221 and-222 (69). AG has been demonstrated to significantly up-regulate anti-fibrotic miR-30a, inhibit pro-fibrotic miR-21, and inhibit miR-206 resulting in increased proliferation of myocytes and aiding in tissue repair (22).

In patients who have DM as a comorbidity with CLI, peripheral neuropathy commonly leads to a delayed clinical presentation of the disease, resulting in severe muscular and vascular damage (75). Several studies have reported that AG administration (300 nmol/kg) ameliorated polyneuropathy in rodent models of diabetic polyneuropathy, evident by increased nerve conduction velocities and temperature sensation (76, 77). Although the mechanisms by which ghrelin alleviates polyneuropathy remain to be identified, these findings further support ghrelin as a potential treatment option for an otherwise intractable disorder. Recently, these findings have also been confirmed in a human study (78), with ghrelin treated patients (1.0 µg/kg for 14 days) having increased motor nerve conduction velocity of the posterior tibial nerve compared with controls. Total symptom score also significantly improved in the treated group compared to controls, suggesting that ghrelin may be a novel therapeutic option for diabetic polyneuropathy. However, a double-blind, placebo-controlled trial is needed in the future.

The results from these studies demonstrate that ghrelin acts to protect against skeletal muscle atrophy, neuropathy, and importantly, aids in muscle regeneration.

The diminished supply of blood to the periphery in CLI and the highly oxidative environment result in severe skeletal muscle damage. SOD-2 is the main antioxidant defence against reactive oxygen species (ROS) production and has been shown to be inhibited in skeletal muscle affected by CLI (79). The two isoforms of ghrelin, AG and Des-AG, have been shown to exert potent anti-inflammatory and antioxidant effects throughout the vasculature.

AG has been shown to exhibit anti-apoptotic effects through the strong activation of Akt and ERK, mediated by the PI3K and MAPK pathways (80), and to inhibit pro-inflammatory cytokine production in vitro through the inhibition of nuclear factor-kappa B (81). AG has been shown to inhibit apoptosis, and blunt ROS production in HUVECs treated with high glucose, replicating diabetic vascular complications, via the PI3K/Akt pathway (82). AG (10⁶ M) inhibits advanced glycation end products-mediated cell apoptosis via the ERK1/2 and PI3K/Akt pathways (83). Moreover, ghrelin has been shown to inhibit vascular oxidative stress through inhibition of vascular NAD(P)H oxidases (84).

Des-acylated ghrelin has been shown to protect against oxidative stress-induced apoptosis through the class III histone deacetylase sirtuin (SIRT1), a nicotinamide adenine dinucleotide dependent histone/protein deacetylase signalling pathway (85). Activation of this pathway has been shown to reduce apoptosis and protects endothelial progenitor cells from ROS-mediated damage under conditions of diabetes (86, 87). In a murine model of CLI and glucose intolerance, Des-AG was also demonstrated to rescue miR-126 expression, a regulator SIRT1 and SOD-2, leading to improved oxidative stress levels and improved recovery (71).

Atherosclerosis underlies most of CLI. Circulating ghrelin concentrations have been shown to be reduced in several atherosclerotic diseases (88–92). To date, the circulating ghrelin levels in patients with PAD or CLI remain unknown. However, in diseases with a similar aetiology, ghrelin levels have been shown to be dysregulated. These include patients with unstable angina (1.04 ± 0.08 ng/mL), acute myocardial infarction (1.07 ± 0.11 ng/mL), and asymptomatic coronary artery disease (2.1 ± 0.8 ng/mL), which have all been shown to have lower total ghrelin concentrations compared to controls (6.2 ± 4.1 ng/mL) (88). In addition to total ghrelin, low circulating levels of Des-AG (~78.2 fmol/mL) have been shown to be associated with increased risk of cardiovascular events in older hypertensive individuals (89). However, further studies are warranted to elucidate the pathogenic mechanisms underlying this association, giving clarity to the clinical value of the measure. In contrast, high plasma concentration of ghrelin has been shown to protect against coronary heart disease (90), and AG is significantly higher in patients with ischemic heart disease (coronary artery disease) compared to controls (valvular heart disease; 32 ± 3 vs. 16 ± 2 pg/mL) (91), potentially demonstrating a role for ghrelin in auto- and paracrine self-protective mechanisms of the ischemic heart. In addition, total circulating ghrelin concentrations are decreased in patients with acute ischemic stroke compared to age- and sex-matched controls (3.47 ± 1.44 vs. 5.93 ± 2.78 ng/mL) (92).

In a murine model of atherosclerosis, AG (10⁻⁹ mol/kg/day for 4 weeks) has also been shown to reduce the formation of atherosclerotic lesions, increase plaque stability, ameliorate activation of endoplasmic reticulum stress, and decrease intima-media thickness (93, 94).

Collectively, this evidence from the literature suggests that plasma ghrelin concentration may be a novel prognostic marker of the extent and severity of various forms of atherosclerotic disease. Additionally, ghrelin may be a promising adjunct therapy for the treatment of CLI and the underlying mechanisms of CLI, atherosclerosis (95).

Vascular calcification is characterized by the progressive enlargement of calcium deposits in the major arteries, and is an independent risk factor for CLI (96). Serum ghrelin level decreases with the severity of the tibial artery stenosis in diabetic patients (mild stenosis, 167.71 ± 16.73; moderate stenosis, 105.72 ± 10.51; severe stenosis/occlusion, 53.11 ± 5.65 ng/mL) (97). In vivo and in vitro models of vascular calcification consistently demonstrate that ghrelin peptide levels and mRNA expression are significantly reduced during calcification. However, treatment with exogenous AG (10⁻⁸–10⁻⁵ mol/L) effectively attenuates the severity of this calcification (97–99). This attenuation is likely mediated through the osteoprotegerin, receptor activator of nuclear factor-kappa B ligand, and the receptor activator of NF-κB axis which regulates vascular calcification.

Combined, it is shown that serum ghrelin may be a novel predictor of vascular calcification in diabetic patients and that exogenous administration of ghrelin may be an effective agent in attenuating calcification.

AG has been shown to induce post-ischemic angiogenesis through the up-regulation of miR-126 (22), the most characterized and abundant pro-angiogenic miR in endothelial cells [reviewed in Ref. (117)]. miR-126 resides within intron seven of the epidermal growth factor-like domain, multiple 7 gene on chromosome 9q34.3, and gives rise to two mature miRs; miR-126-3p and miR-126-5p. miR-126-3p has been well studied for its role in vascular remodeling [reviewed in Ref. (133)]. miR-126 is essential for blood vessel growth in zebrafish (118) and mice (119). Specific deletion of miR-126 is 50% embryonic lethal, with surviving mutants expressing fragile, leaky vessels, and severe haemorrhage (120, 134). These findings highlight the fundamental role of miR-126 in regulation of vascular integrity and angiogenesis. Consequently, this fundamental role of miR-126 in the vasculature has led to research investigating its regenerative potential, which was confirmed in several animal models of ischemic vascular injury (135, 136, 137).

Mechanistically, miR-126 targets sprouty-related protein 1 (SPRED1) and phosphoinositol-3-kinase regulatory subunit 2 (PIK3R2/p85β), both of which suppress VEGF, via PI3K and Akt signalling pathways, respectively. Thus, miR-126 promotes VEGF expression by targeting several pathways, which promotes an angiogenic response (118).

Des-acylated ghrelin has been shown to exhibit an atheroprotective effect by restoring miR-126 expression. In a murine model of CLI, Des-AG administration increased miR-126, which increased SIRT1 expression and prevented cell senescence via reduced p53 and H3K56 acetylation, protecting deoxyribonucleic acids from damage (71, 138). Furthermore, Des-AG induced an anti-adhesive and anti-inflammatory endothelial phenotype, via miR-126 regulated VCAM-1 post-transcriptional regulation (71). Finally, Des-AG administration also restored SOD-2 expression by diminishing ROS production (71).

MicroRNA-206 is an evolutionally conserved miR, sharing common expression in muscle from Caenorhabditis elegans to human (122). miR-206 is also similar to miR-1, only differing by four nucleotides as mature miRs. miR-206 is found in the intergenic region on chromosome 6p12.2 and has been proposed to be a negative regulator of angiogenesis. This is shown to be through the modulation of the potent angiogenic factor VEGF (122, 123). Further, miR-206 has been shown to specifically suppress VEGF expression in several types of cancer and smooth muscle cells (140), clearly demonstrating the role of miR-206 as a negative regulator of angiogenesis. AG has been shown to suppress miR-206 expression, leading to increased VEGF expression and angiogenesis (22). This indicates that AG up-regulates several pro-angiogenic miRs and down-regulates antiangiogenic miRs leading to a pro-angiogenic environment.

MicroRNA-92a is a conserved endothelial cell-specific miR, in the miR-17~92 cluster located at chromosome 13q31.3 (124). miR-92a inhibits endothelial cell sprouting and neovascularization following ischemia, through targeting of the protective endothelial genes integrin α5 and SIRT1 (124, 141). Inhibition of miR-92a has been shown to improve angiogenesis and recovery in murine models of chronic ligation of the anterior descending coronary artery (124), CLI (124), vascular injury (142), and in a porcine model of ischemia/reperfusion injury (143).

Exogenous AG treatment has also shown to decrease miR-92a, which negatively modulates integrin α5, which is crucial for the activation of Akt (144). This leads to improved cell survival through the anti-apoptotic factor B cell lymphoma 2 (145) and improved angiogenesis. Combined, ghrelin’s ability to reduce miR-92a expression leads to further regulation of integrin α5 and SIRT1, both of which critically influence endothelial cell proliferation and migration (146).

The miR-221/-222 cluster is encoded by a highly conserved gene cluster on the human Xp11.3 chromosome (147). miR-221/-222 have been shown to control the differentiation and maturation of skeletal muscle cells, by modulating the protein levels of the cell-cycle inhibitors p27 and p57 (126). Des-AG has been shown to induce skeletal muscle regeneration via SOD-2 expression and miR-221/-222 driven post-transcriptional regulation of the Cip/Kip family members P27^KIP1 (also known as CDKN1B) and P57^KIP2 (also known as CDKN1C) (127), leading to satellite cell differentiation and myofiber regeneration (69). This indicates Des-AG may benefit CLI in that it can up-regulate miR-221/-222, leading to skeletal muscle regeneration. However, miR-221/-222 inhibits endothelial cell proliferation and motility, thus blocking angiogenesis by targeting the pro-angiogenic c-kit (70, 147), suggesting that Des-AG may improve the skeletal muscle recovery following CLI, but at the same time may have detrimental effects on the neovasculature.

MicroRNA-30a is a member of the miR-30 family, located on chromosome 6q13 (148). miR-30a has recently been shown to play a fundamental role in myocardial (129), peritoneal (130), and hepatic fibrosis (149) through the negative regulation of its target protein Snail 1. AG has been shown to increase miR-30a in a model of CLI, leading to diminished skeletal muscle fibrosis and improved recovery (22).

MicroRNA-21 is a well characterized pro-fibrotic miR [reviewed in Ref. (150)], located on chromosome 17q23.2, within an intron of the transmembrane protein 49 gene (151). miR-21 promotes fibrosis in several tissues including the heart (132, 151), kidneys (150), and skeletal muscle (152) through fibroblast proliferation by activation of ERK–MAPK pathway (131). Sprouty homolog 1 (SPRY1), a potent inhibitor of the Ras/MEK/ERK pathway, has been shown to be a direct target of miR-21 (131, 153). Thus, miR-21 mediates ERK–MAP kinase activity by means of an effect on SPRY1, leading to fibrosis. Moreover, phosphatase and tensin homolog (PTEN) a well-established target of miR-21, is involved in fibrosis by stimulation of the epithelial-to-mesenchymal transition (132) and interrupts the down- stream activation of Akt, a regulator in fibrotic diseases. Pro-fibrotic TGF-β signalling, which contributes to the progression of fibrosis by promoting fibroblasts activation, has been shown to be regulated by miR-21 via a PTEN/Akt-dependent pathway (154). AG has been demonstrated to reduce miR-21 in a model of CLI, thus reducing the TGF-β induced fibrotic signalling cascade (22), leading to decreased fibrosis and improved recovery.

AG and Des-AG exhibit pleiotropic beneficial effects, which all may contribute to improved recovery following CLI. The influence of ghrelin on miRs sheds light on the molecular mechanisms that underpin ghrelin’s therapeutic actions.