CGRP

Chapter 189

CGRP Ross King and Susan D. Brain

ABSTRACT Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that belongs to a family of structurally related peptides, including adrenomedullin (AM), adrenomedullin II (AM2, or intermedin), and amylin (AMY). CGRP expression is largely concentrated within small-diameter primary sensory afferents that innervate the vasculature. When released into and around the vasculature, CGRP acts as a potent vasodilator. To achieve its effect, CGRP binds to a Class B G protein-coupled receptor, known as the calcitonin receptor-like receptor (CLR), which in turn is coupled to a receptor activity modifying protein 1 (RAMP1) to confer specificity for the CGRP ligand. Abnormal CGRP activity has been identified as a potential causative factor within the pathophysiological setting of migraine and, as such, antagonists of the CGRP receptor are in development for the treatment of this disease. Given its potent activity and ability to be released throughout the body, CGRP has the potential to maintain cardiovascular homeostasis, warranting future work to fully characterize its effects.

DISCOVERY Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that was first identified in 1982 by Amara et al. It was discovered in the thyroid tissue of aging rats, where alternative processing of RNA transcripts from the calcitonin gene was shown to result in the production of distinct mRNAs encoding CGRP. Calcitonin mRNA comprises exons 1, 2, 3, and 4 of the calcitonin gene, whereas CGRP mRNA comprises exons 1, 2, 3, 5, and 6, as shown in Fig. 1. Expression of calcitonin mRNA was shown to be largely confined to the thyroid tissue, while CGRP-specific mRNA is considered to predominate throughout the sensory nervous system.2 After this discovery, a human form of CGRP was isolated from medullary thyroid carcinomas.22 Two distinct isoforms of CGRP are now known to exist, termed αCGRP and βCGRP. αCGRP is formed from the alternative splicing of mRNA transcribed from the calcitonin gene located on chromosome 11, whereas the beta isoform is transcribed from its own gene located on the same chromosome.1 Because of the similar organization 1394

of both genes, it is believed that the two separate genes arose by duplication. Both isoforms of the mature peptide are structurally similar, with the β isoform only differing by 3 amino acid residues in humans and 1 amino acid in rodents. Indeed, both isoforms display comparable biological activity and primarily differ in their tissue distribution. αCGRP is most commonly found within peripheral sensory neurons, whereas βCGRP seems to be more restricted to nerves of the enteric plexuses. Little is known about the specific function of the β isoform, and thus from this point, only αCGRP will be discussed.

DISTRIBUTION OF THE mRNA/PEPTIDE IN THE CARDIOVASCULAR SYSTEM Neuronal Localization of CGRP CGRP is widely distributed throughout the central and peripheral nervous systems. It is primarily located within unmyelinated, small-diameter sensory C, and thinly myelinated Aδ fibers that are commonly found in close contact with the vasculature, although with a greater degree of innervation on the arterial side of the circulation. Sensory nerve terminals have been shown to protrude as far into the vascular wall as the adventitial–medial border, enabling a direct influence over vascular function. A well-established mechanism by which CGRP and other neuropeptides are released from sensory nerves is after neuronal stimulation with the potent chilli-pepper extract, capsaicin. Capsaicin is a highly selective agonist for the vanilloid subtype of transient receptor potential class of ion channel (TRPV1), and its binding to TRPV1 induces the opening of a central pore that is permeable to the influx of extracellular cations, with a degree of selectivity for divalent ions, such as calcium. An increase in intracellular calcium leads to depolarization of the neuronal membrane and triggering of calcium-dependent exocytotic mechanisms and release of CGRP. Indeed, long-term treatment of rodent neonates with capsaicin leads to neuronal excitotoxicity and subsequent sensory nerve loss. Other activators of TRPV1 include ­noxious heat and the high concentrations of protons associated with lowered Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00189-5 Copyright © 2013 Elsevier Inc. All rights reserved.

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CALC I gene Exon 1

Exon 2

Exon 3

Exon 4

Calcitonin mRNA

Exon 5

Exon 6

CGRP mRNA

FIGURE 1  Arrangement of CALC I exons encoding either calcitonin or CGRP mRNA.

environmental pH.28 There has been recent research involving other TRP receptors, one of which is the ankyrin receptor TRPA1. This is found on 50% of sensory nerves and when stimulated is capable of releasing vasodilator CGRP, depending on the circumstance.24 Physiological plasma levels of CGRP in humans are usually within the low picomolar range, and this low level is generally attributed to overspill from the site of release, as opposed to having a systemic circulatory effect. Once released, it is unclear as to how CGRP may be metabolized to an inactive form, and several removal mechanisms are likely to take place.4 Mast cell tryptase and other peptidases have been shown to be capable of breaking down CGRP at extravascular sites, and there also exists a role for a matrix metalloprotease II enzyme in CGRP metabolism. Furthermore, there is some evidence to suggest that CGRP is taken up by the nerve terminal following its repolarization, where it may be recycled for reuse or marked for degradation.

Novel, Nonneuronal Sites of CGRP Expression Aside from the canonical synthesis and release of CGRP from perivascular sensory neurons, other nonneuronal sources of the peptide are slowly beginning to emerge. A range of studies have been performed to investigate endothelial progenitor cell (EPC) function, and this cell type has been shown to actively express and release CGRP.11 Moreover, it has been suggested that mature endothelial cells (ECs) possess the ability to express both isoforms of CGRP and release CGRP in a TRPV1-dependent manner. This endothelial-derived CGRP has been suggested to play an important role in the protection of endothelial function induced by acute heat stress.31 Furthermore, a study published by Wang et  al. in 2002 suggests that human lymphocytes constitutively express both isoforms of CGRP,

although this finding is yet to be confirmed in subsequent experiments.30

RECEPTORS AND SIGNALING CASCADES Our knowledge of the CGRP receptor and its associated signaling cascades has increased in a short span of time. Historically, two distinct receptors for CGRP were initially believed to exist: the CGRP1 and CGRP2 receptors. The idea of heterogeneous CGRP receptor subpopulations was put forward when it was discovered that responses to CGRP in the guinea pig atria could be blocked by the peptide antagonist, CGRP8–37, whereas in the rat vas deferens, this fragment was less capable of antagonizing CGRP-induced effects.9 However, by using molecular and pharmacological techniques, we can now appreciate that apparent differences in CGRP activity may be ascribed to the activation of receptors for other members of the calcitonin family, such as adrenomedullin and amylin.13 The identity of the CGRP receptor takes the form of a multiprotein oligomer comprising a secretin-like (“Class B”) G protein-coupled receptor (GPCR), known as the calcitonin receptor-like receptor (CLR), bound to a receptor activity modifying protein (RAMP)1 molecule. Dimerization of the CLR to other RAMPs confers pharmacological activity on other members of the calcitonin family of peptides: RAMP2 and RAMP3 association leads to the formation of AM1 and AM2 receptors, respectively.29 Fig. 2 illustrates how all CGRP receptor protein subunits combine to form a fully functional receptor. Assembled CGRP receptors are known to be expressed in a variety of cell types throughout the cardiovascular system, including vascular smooth muscle cells (VSMCs), endothelial cells, cardiac myocytes, some populations of leukocytes, and on the cell bodies of perivascular sensory neurons.

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FIGURE 2  Schematic representation of the oligomerization of CGRP receptor components. The CLR binds to RAMP1 to confer ligand specificity to CGRP. CGRP receptor activation typically results in the activation of a G protein, usually Gαs. The RCP positively regulates Gαs-mediated signal transduction.

Critical to the advancing of our insight into receptor function and into the formation of novel therapeutic strategies is the development of a “pharmacological toolkit,” including antagonists selective for distinct receptor subpopulations. The first antagonist for the CGRP receptor was developed with the finding that truncation of the first seven amino acid residues of the peptide (resulting in the CGRP8–37 peptide fragment) binds to the receptor complex without activating signaling pathways, thereby competing with the full-length peptide for receptor occupancy.6 However, in keeping with its peptide nature, CGRP8–37 is not an ideal tool for interrogating specific signaling pathways. More recently, however, novel nonpeptide antagonists have been designed to selectively target the interface between CLR and RAMP1, binding to the CLR at methionine-42 and RAMP1 at tryptophan-74, and these have been shown to have considerable efficacy in the treatment of migraine-associated headache: olcegepant (BIBN4096; 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-d-tyrosyl]-1-lysyl]4-(4-pyridinyl)piperazine) and telcagepant (MK-0974; [N-[(3R,6S)-6-(2.3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5b]pyridin-1-yl)piperidine-1-carboxamide]).21

CGRP Receptor Components The main functional component of the CGRP receptor critical for signal transduction following agonist binding is the CLR. Consistent with the highly conserved structure of GPCRs, the CLR contains regions that are critical for GPCR signaling. The CLR possesses a large N-terminal extracellular domain

(ECD), three extracellular loops (ECLs), seven transmembrane (TM) domains, three intracellular loops (ICLs) and an intracellular C-terminus. The manner in which peptide ligands bind to Class B GPCRs, including the CLR/RAMP1 complex, is generally described as the “two-domain” model. In this model, the C-terminus of the peptide binds with high affinity to the N-terminal ECD and is subsequently delivered to the ECL and extracellular TM domains of the receptor while also binding to the RAMP molecule, resulting in the activation of the receptor by inducing a change in receptor conformation.15 The CLR alone is incapable of responding to CGRP stimulation and requires the heterodimerization of the single transmembrane-spanning RAMP1 to offer full functionality. Indeed, association of RAMP1 with the CLR is critical for the maturation of the CLR via N-terminal glycosylation and subsequent export of the receptor from the endoplasmic reticulum to the plasma membrane, where it is able to interact with its peptide agonist.19 Coupling of the CLR to RAMP1 produces a receptor that is responsive to CGRP and is blocked by BIBN4096BS/CGRP8–37. Association of the CLR with RAMP2 produces a receptor for AM that is blocked by its associated peptide fragment AM22–52, and binding of RAMP3 produces a receptor responsive to both CGRP and AM, about which very less is known. In addition to RAMP1, It is now known that a small intracellular peripheral membrane protein also associates with the CLR, as shown by coimmunoprecipitation studies. This receptor coupling protein (RCP) is important for the amplification of signal generated by CGRP receptor activation. Endogenously expressed by most cell types, molecular knockdown techniques have shown that ablation of RCP activity leads to a decreased production of cAMP secondary to CGRP receptor activation but not from other receptor systems that are coupled to Gαs. Therefore, it seems that the association of RCP with the CLR is important for the regulation of efficient intracellular signal transduction.10

CGRP Receptor Signaling Activation of the CGRP receptor is typically associated with an increase in the formation of the intracellular second messenger molecule cyclic 3′-5′-adenosine monophosphate (cAMP), as a result of the small G protein Gαs associating with the CLR and promoting adenylyl cyclase activity. Accumulation of cAMP within subcellular signaling compartments results in the temporospatial activation of the cAMP-dependent protein kinase (PKA), which itself is capable of targeting multiple protein substrates for phosphorylation, thereby altering their functionality. PKAdependent events occurring downstream from CGRP receptor stimulation have been shown to include alterations of the activity of critical signaling proteins pertaining to cell excitability, such as ATP-sensitive potassium channels and

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L-type calcium channels, and DNA transcription as a result of the activation of the cAMP response element binding protein (CREB). Furthermore, consistent with other members of the GPCR superfamily, it is likely that the CGRP receptor may couple to multiple additional signaling networks, such as other G proteins including Gi/o and Gq/11. In addition, there is a growing body of evidence to suggest that CGRP receptor stimulation may result in the activation of mitogen-activated protein kinases (MAPKs), which are essential to cell proliferation and survival. It is yet unclear how the receptor might signal to these kinases, though a possible mechanism may be through β-arrestin-mediated signaling; β-arrestin has been shown to associate with the CGRP receptor and facilitates receptor desensitization and internalization. Therefore, it is plausible that the CGRP receptor may transmit a signal in this manner, as it has been shown with other receptor systems.29 To be able to maintain efficient control of CGRP activity, the signal generated by the receptor must be terminated in a suitable manner. Although Class B GPCRs are less well characterized than Class A GPCRs, it is known that they both share the ability to undergo agonist-induced homologous desensitization, and this has indeed shown to be the case for the CGRP receptor. In cultured human neuroblastoma cells and VSMCs, pretreatment of cells with CGRP for 20min resulted in an attenuated elevation of cAMP on a secondary exposure. It has since been shown that after the binding of CGRP to its cognate receptor, the CLR undergoes rapid phosphorylation and binds β-arrestin where it is thereby internalized as part of a ternary complex comprising the CLR, RAMP1, and β-arrestin molecules. The association of β-arrestin with the receptor complex is stable and long lasting, showing an internalization profile similar to that of the V2 vasopressin receptor.12

BIOLOGICAL ACTIONS WITHIN THE CARDIOVASCULAR SYSTEM CGRP may have a variety of roles within the cardiovascular system, but the feature that is best characterized is its ability to act as a potent vasodilator. Often considered the most potent microvascular vasodilator to date, its property was first discovered by Brain et al. in 1985,3 where intradermal injections of femtomolar concentrations of CGRP into the human forearm was sufficient to induce surface erythema of the skin, attributable to local vasodilatation and, as a result, increased blood flow. Higher doses of CGRP were found to produce a reddening of the skin that lasted for several hours.3 Intravenous delivery of CGRP into rodents causes systemic hypotension coupled with a positive inotropic and chronotropic response from the heart, though these effects are considered to be secondary to stimulation of the sympathetic nervous system as part of the baroreceptor reflex,

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rather than a direct action of CGRP on the heart. The vasodilator activity of CGRP is predominantly mediated by the binding of CGRP to the CGRP receptor complex embedded within the plasma membrane of VSMCs and, to an extent, the ECs lining the blood vessels. Within the VSMC, stimulation of the CGRP receptor leads to an increase in the formation of cAMP and subsequent activation of PKA, which has been shown to target the ATP-sensitive potassium channel for phosphorylation, leading to cellular relaxation as a result of membrane hyperpolarization. Alternatively, CGRP may interact with receptors located on ECs, leading to the downstream phosphorylation and activation of endothelial nitric oxide synthase (eNOS) and liberation of nitric oxide (NO) from the EC, which is then capable of diffusing to the VSMC layer and causing relaxation via the stimulation of soluble guanylate cyclase and triggering an accumulation of intracellular cGMP as a result.4

CGRP and Control of Blood Pressure Although it is evident that CGRP possesses clear vasomotor activity, it is less clear as to what sort of effect it has on hemodynamics under normal physiological conditions. A range of studies has been performed using murine knockout models to address this question, with some conflicting results. Some groups report no change in baseline blood pressure in mice with the removal of αCGRP, whereas others have witnessed a significant increase in blood pressure in its absence. Difficulty arises when interpreting these findings, however, as the generation of CGRP knockout mice can differ between groups, where some animals have the complete CALC I gene removed (thereby preventing both calcitonin and αCGRP synthesis) and others lack only αCGRP. It is worth noting, however, that mice holding the sole deletion of CGRP seem to be more likely to have normal resting blood pressures.27 Adding further credence to the hypothesis that CGRP plays a minor role in baseline blood pressure regulation, studies performed in a variety of species have shown that injection of the CGRP receptor antagonist CGRP8–37 or CGRP receptor-directed antibodies has no effect on systemic blood pressure. Furthermore, injection of the selective CGRP receptor antagonist BIBN4096BS has also been shown not to exert an effect on resting heart rate or blood pressure across a range of species, including humans.23 Therefore, it may be the case that the functional consequences of CGRP are more pronounced within the microcirculation of specific tissues, as opposed to the entire circulation. This is supported by findings that indicate that administration of CGRP at doses below those required to produce systemic hypotension produces facial flushing, indicative of a specific sensitivity to CGRP within the cutaneous microcirculation. Indeed, capsaicin-induced denervation of peripheral sensory nerves in neonatal rats

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typically results in the formation of skin lesions resulting from the removal of neuropeptidergic control of the dermal microcirculation. Furthermore, skin flap survival in sensory nerve-depleted rats is reduced when compared with those with sensory nerves intact and skin flap survival can be rescued with the administration of CGRP. In mice subjected to hind limb ischemia, neovascularization is significantly impaired after blockade of CGRP. These findings are in keeping with the principal role of CGRP as a regulatory microvascular neuropeptide, being released from perivascular sensory nerves at specific sites to produce local vascular control as opposed to behaving in an endocrine manner.4

Cardiac Effects of CGRP The role of CGRP within cardiac physiology is less clear. CGRP-immunoreactive fibers have been shown to innervate regions of the myocardium and the coronary vessels that supply the heart with blood. A range of studies has been performed that propose a cardioprotective role for CGRP. Isolated heart preparations using mice with mixed CGRP/ calcitonin gene deletion were found to have an increased susceptibility to ischemia/reperfusion injury, associated with a marked increase in markers of myocardial cell damage, coupled with an increase in reactive oxygen species generation. Recovery of coronary flow after acute myocardial ischemia was also significantly impaired in mice lacking CGRP. Similar results were obtained with pharmacological blockade of the CGRP receptor with CGRP8–37.17 More recently, it has been shown that mice subjected to streptozotocin-induced diabetes mellitus have an impaired ability to produce cardioprotection via ischemic preconditioning, and this is attributed to a loss of CGRP activity. This effect was rescued by CGRP adenoviral gene delivery and, interestingly, post-IR myocardial function was also improved in nondiabetic hearts receiving CGRP gene transfer.34 Furthermore, in cultured rat cardiomyocytes, CGRP was shown to inhibit the accelerated apoptosis of myocardial cells induced by norepinephrine.32 This finding may have particular relevance to research into heart failure, as patients often present with an increased activity of the sympathetic nervous system and increased catecholamine release. Although the cardiac effects of CGRP are not as well documented as those in the vasculature, it is conceivable that increasing cardiac CGRP activity by pharmacological or genetic means may represent a new way to prevent the deleterious effects of ischemia/reperfusion injury and associated cell death.

PATHOPHYSIOLOGICAL IMPLICATIONS Migraine Despite affording apparent protective effects within the cardiovascular system, CGRP has also been shown to play a

key role in the pathophysiology of migraine. Migraine is a debilitating cerebrovascular condition affecting around 15% of people, characterized by a headache comprising a unilateral throbbing sensation with a range of associated symptoms. Migraine may manifest in an episodic or chronic manner, with both instances impacting in a detrimental manner on society and the patient. Current standard treatment for the symptoms of migraine is generally limited to the triptan family of drugs, which possess dual pharmacological activity at 5-HT1B/1D receptors. However, because of their ability to constrict blood vessels, the use of triptans is contraindicated in patients with cardiovascular complications, such as hypertension and coronary artery disease.14 Interestingly, pharmacological treatment of migraine now seems to be moving in the direction of CGRP receptor antagonism. Soon after the discovery of CGRP, early localization studies showed its presence in cranial blood vessels and in the trigeminal peripheral neurons that innervated this region of the vasculature, where it seemed to function as a protective neurotransmitter that exerted reflex control over hypercontractile vessels. Experiments were also performed in human patients suffering from migraine, in whom collection of blood from the external jugular vein draining the extracerebral tissues was analyzed and found to contain elevated concentrations of CGRP (92 pM versus 40 pM in healthy control subjects), but not of other neuropeptides. Moreover, intravenous delivery of CGRP was shown to dilate cerebral arteries and arterioles concomitant with an increased incidence of migraine-like headache. One theory suggests that the pain associated with migraine is a result of the extracerebral blood vessels (e.g., the middle meningeal artery and its dural arterioles) that supply the dura mater relaxing to an exaggerated extent and thereby stimulating the perivascular nociceptive neurons. These observations together led to the rational development of the nonpeptide CGRP receptor antagonists: olcegepant (BIBN4096BS, administered intravenously) and telcagepant (MK-0974, given orally). Owing to its oral availability, telcagepant has superseded olcegepant in the treatment of migraine.14

Raynaud’s Phenomenon Another clinical problem that may benefit from manipulation of the CGRP system is Raynaud’s phenomenon. Characterized by inappropriate vasoconstriction and pain within the cutaneous microcirculation (commonly affecting the digits of the hand), the syndrome can restrict adequate tissue perfusion, leading to ischemia and potentially the development of gangrene. Patients suffering from Raynaud’s have been shown to have increased levels of the potent pressor agent, endothelin, and decreased levels of CGRP within the digital circulation. Moreover, patients receiving CGRP infusion at room temperature were found to respond in a

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manner similar to that of healthy control subjects. However, at cold temperatures (5 °C), patients were shown to exhibit a significantly attenuated response to CGRP infusion.5 Therefore, the disease may present as a result of increased responsiveness to endothelin and a lack of responsiveness to CGRP, opening a potential avenue for CGRP receptor agonists in the treatment of the disease. Indeed, recent in vivo and ex vivo studies have shown that CGRP is capable of terminating long-lasting vasopressor responses to endothelin, further strengthening a potential therapeutic role for CGRP within this setting.20

Hypertension and Vascular Inflammation Finally, there is developing evidence to show that CGRP activity seems to become more important in resisting the onset of hypertension and its associated vascular dysfunction within several experimental models. Indeed, it has recently been shown that global overexpression of RAMP1 in mice results in a significant attenuation of ATII-induced hypertension and improved baroreceptor reflex activity.26 Similarly, studies performed with the peptide CGRP receptor antagonist CGRP8–37 have shown that blockade of the CGRP receptor exaggerates cardiovascular pathologies induced by hypertensive stimuli, such as salt-induced hypertension and l-NAME–induced hypertension.27 Moreover, although plasma CGRP concentrations in humans are generally considered to be low, there is evidence to suggest that circulating levels of CGRP can become reduced in patients with essential hypertension. These clinical findings are supported by rodent studies, which have shown that CGRP release and activity may be reduced when levels of angiotensin (AT) are raised. Furthermore, in spontaneously hypertensive rats (SHR—considered a relevant model of essential human hypertension), the level of CGRP mRNA expression by the dorsal root ganglion cells (the principal site of CGRP synthesis) has been shown to decline with age. Therefore, the development of hypertension and associated vasculopathy may be linked to a decrease in CGRP activity within the cardiovascular system. Indeed, there is some evidence to suggest that chronic supplementation of the traditional Chinese medicine and TRPV1 agonist rutaecarpine may have an antihypertensive effect that is attributable to increased CGRP release into the systemic circulation.8 This protective effect has been documented in hypertension induced by renal phenol injury, two-kidney, one-clip surgery, and in spontaneously hypertensive rats. Additionally, CGRP released from TRPV1 after rutaecarpine stimulation was demonstrated to have a beneficial effect in reducing platelet aggregation by inhibiting the release of platelet-derived tissue factor.18 Therefore, targeting of TRPV1 with agonists such as rutaecarpine may provide a valid strategy for the treatment of hypertension and its associated conditions.

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Hypertension often copresents as an increase in systemic blood pressure and also extensive vascular remodeling and dysfunction. This commonly includes an accelerated rate of hypertrophy and hyperplasia of the VSMCs within the medial wall of the blood vessel. Furthermore, endothelial dysfunction is known to occur, typically characterized by an impaired capability of the endothelial cell to produce the vasoactive mediator, NO. Transgenic overexpression of the RAMP1 component of the CGRP receptor has been shown to protect against ATII-induced endothelial dysfunction, measured by the ability of isolated vessels to relax in response to acetylcholine stimulation of the endothelium to produce NO.7 Critical to the repair of damaged regions of the endothelium are circulating EPCs: bone marrowderived circulating cells that are now appreciated to play an important role in vascular repair after injury and also neovascularization. It is believed that a decline in blood EPC content and activity could predispose patients toward future cardiovascular events. CGRP is known to be synthesized and secreted by this cell type, and it has been shown that EPC-derived CGRP can protect against acceleration of EPC senescence induced by ATII and downstream production of oxygen radicals.35 EPC-derived CGRP also seems to attenuate the degree of VSMC hypertrophy within an ATII model of hypertension.11 This finding is strengthened by in vitro studies examining ATII-induced VSMC proliferation and how this activity can be inhibited by exogenous CGRP application.25 Furthermore, CGRP-expressing EPCs have been shown to be of some therapeutic value in a model of pulmonary hypertension, where animals receiving CGRPpositive EPCs had a lowered vascular resistance within the pulmonary circulation and decreased smooth muscle thickening.33 Another characteristic associated with hypertension is an exacerbated incidence of vascular inflammation. Huang et  al. showed that exposure of activated microvascular ECs to CGRP significantly attenuated the ratio of inflammatory cells such as neutrophils and monocytes migrating through the endothelial monolayer. This was attributed to a decrease in chemokine expression by the endothelial cell following CGRP stimulation, as a result of a reduced activation of the proinflammatory transcription factor, NFκB.16 Chronic activation of NFκB is known to predispose arteries to developing atherosclerotic lesions. As interactions between circulating leukocytes and the vasculature are important factors in the etiology of inflammatory diseases such as atherosclerosis, there may be some benefit in manipulating the CGRP signaling pathway to prevent atherogenesis and related inflammatory conditions within the vasculature. In summary, it is now appreciated that CGRP has a wide range of effects in the cardiovascular system and may be involved in multiple pathophysiological circumstances. It is probable that we will witness the progression of CGRP

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receptor antagonists for the treatment of migraine into the clinic within the coming years. With particular interest to more prevalent and serious cardiovascular diseases, however, it is possible that CGRP plays an important protective role in the pathophysiology of hypertension and its associated vascular disease component. Therefore, future basic and clinical research may lead us in the direction of developing ways by which CGRP activity may be selectively stimulated to develop novel treatments for cardiovascular conditions.

REFERENCES 1. Alevizaki M, Shiraishi A, Rassool FV, Ferrier GJ, MacIntyre I, Legon S. The calcitonin-like sequence of the beta CGRP gene. FEBS Lett 1986;206:47–52. 2. Amara SG, Jonas V, Rosenfeld MG, Ong ES, Evans RM. Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 1982;298:240–4. 3. Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature 1985;313:54–6. 4. Brain SD, Grant AD. Vascular actions of calcitonin gene-related peptide and adrenomedullin. Physiol Rev 2004;84:903–34. 5. Bunker CB, Goldsmith PC, Leslie TA, Hayes N, Foreman JC, Dowd PM. Calcitonin gene-related peptide, endothelin-1, the cutaneous microvasculature and Raynaud’s phenomenon. Br J Dermatol 1996;134:399–406. 6. Chiba T, Yamaguchi A, Yamatani T, Nakamura A, Morishita T, Inui T, et al. Calcitonin gene-related peptide receptor antagonist human CGRP(8-37). Am J Physiol 1989;256:E331–5. 7. Chrissobolis S, Zhang Z, Kinzenbaw DA, Lynch CM, Russo AF, Faraci FM. Receptor activity-modifying protein-1 augments cerebrovascular responses to calcitonin gene-related peptide and inhibits angiotensin II-induced vascular dysfunction. Stroke 2010;41:2329–34. 8. Deng P-Y, Ye F, Cai W-J, Tan G-S, Hu C-P, Deng H-W, et al. Stimulation of calcitonin gene-related peptide synthesis and release: mechanisms for a novel antihypertensive drug, rutaecarpine. J Hypertens 2004;22:1819–29. 9. Dennis T, Fournier A, Cadieux A, Pomerleau F, Jolicoeur FB, St Pierre S, et al. hCGRP8-37, a calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery. J Pharmacol Exp Ther 1990;254:123–8. 10. Evans BN, Rosenblatt MI, Mnayer LO, Oliver KR, Dickerson IM. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J Biol Chem 2000;275:31438–43. 11. Fang L, Chen M-F, Xiao Z-L, Liu Y, Yu G-L, Chen X-B, et al. Calcitonin gene-related peptide released from endothelial progenitor cells inhibits the proliferation of rat vascular smooth muscle cells induced by angiotensin II. Mol Cell Biochem 2011. 12. Hay DL, Poyner DR, Smith DM. Desensitisation of adrenomedullin and CGRP receptors. Regul Pept 2003;112:139–45. 13. Hay DL, Poyner DR, Quirion R, International Union of Pharmacology. International Union of Pharmacology. LXIX. Status of the calcitonin gene-related peptide subtype 2 receptor. Pharmacol Rev 2008;60:143–5. 14. Ho TW, Edvinsson L, Goadsby PJ. CGRP and its receptors provide new insights into migraine pathophysiology. Nat Rev Neurol 2010;6:573–82.

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15. Hoare SRJ. Mechanisms of peptide and nonpeptide ligand binding to Class B G protein-coupled receptors. Drug Discov Today 2005;10:417–27. 16. Huang J, Stohl LL, Zhou X, Ding W, Granstein RD. Calcitonin gene-related peptide inhibits chemokine production by human dermal microvascular endothelial cells. Brain Behav Immun 2011;25: 787–99. 17. Huang R, Karve A, Shah I, Bowers MC, Dipette DJ, Supowit SC, et  al. Deletion of the mouse alpha-calcitonin gene-related peptide gene increases the vulnerability of the heart to ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2008;294:H1291–7. 18. Li D, Peng J, Xin H-Y, Luo D, Zhang Y-S, Zhou Z, et al. Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats. Peptides 2008;29:1781–8. 19. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998;393:333–9. 20. Meens MJPMT, Mattheij NJA, Nelissen J, Lemkens P, Compeer MG, Janssen BJA, et  al. Calcitonin gene-related peptide terminates longlasting vasopressor responses to endothelin 1 in vivo. Hypertension 2011;58:99–106. 21. Miller PS, Barwell J, Poyner DR, Wigglesworth MJ, Garland SL, Donnelly D. Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74. Biochem Biophys Res Commun 2010;391:437–42. 22. Morris HR, Panico M, Etienne T, Tippins J, Girgis SI, MacIntyre I. Isolation and characterization of human calcitonin gene-related peptide. Nature 1984;308:746–8. 23. Olesen J, Diener H-C, Husstedt IW, Goadsby PJ, Hall D, Meier U, BIBN 4096 BS Clinical Proof of Concept Study Group, et  al. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N Engl J Med 2004;350: 1104–10. 24. Pozsgai G, Bodkin JV, Graepel R, Bevan S, Andersson DA, Brain SD. Evidence for the pathophysiological relevance of TRPA1 receptors in the cardiovascular system in vivo. Cardiovasc Res 2010;87:760–8. 25. Qin X-P, Ye F, Hu C-P, Liao D-F, Deng H-W, Li Y-J. Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells. Eur J Pharmacol 2004;488:45–9. 26. Sabharwal R, Zhang Z, Lu Y, Abboud FM, Russo AF, Chapleau MW. Receptor activity-modifying protein 1 increases baroreflex sensitivity and attenuates angiotensin-induced hypertension. Hypertension 2010;55:627–35. 27. Smillie S-J, Brain SD. Calcitonin gene-related peptide (CGRP) and its role in hypertension. Neuropeptides 2011;45:93–104. 28. Tominaga M, Tominaga T. Structure and function of TRPV1. Pflugers Arch 2005;451:143–50. 29. Walker CS, Conner AC, Poyner DR, Hay DL. Regulation of signal transduction by calcitonin gene-related peptide receptors. Trends Pharmacol Sci 2010;31:476–83. 30. Wang H, Xing L, Li W, Hou L, Guo J, Wang X. Production and secretion of calcitonin gene-related peptide from human lymphocytes. J Neuroimmunol 2002;130:155–62. 31. Ye F, Deng P-Y, Li D, Luo D, Li N-S, Deng S, et  al. Involvement of endothelial cell-derived CGRP in heat stress-induced protection of endothelial function. Vascul Pharmacol 2007;46:238–46.

SECTION | XIV  Handbook of Biologically Active Peptides: Cardiovascular Peptides

32. Zhao F-P, Guo Z, Wang P-F. Calcitonin gene related peptide (CGRP) inhibits norepinephrine induced apoptosis in cultured rat cardiomyocytes not via PKA or PKC pathways. Neurosci Lett 2010;482:163–6. 33. Zhao Q, Liu Z, Wang Z, Yang C, Liu J, Lu J. Effect of preprocalcitonin gene-related peptide-expressing endothelial progenitor cells on pulmonary hypertension. Ann Thorac Surg 2007;84: 544–52.

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34. Zheng L-R, Han J, Yao L, Sun Y-L, Jiang D-M, Hu S-J, et  al. Up-regulation of calcitonin gene-related peptide protects streptozotocin-induced diabetic hearts from ischemia/reperfusion injury. Int J Cardiol 2011. http://dx.doi.org/10.1016/j.ijcard.2011.04.009. 35. Zhou Z, Hu C-P, Wang C-J, Li T-T, Peng J, Li Y-J. Calcitonin generelated peptide inhibits angiotensin II-induced endothelial progenitor cells senescence through up-regulation of klotho expression. ­Atherosclerosis 2010;213:92–101.