Sage Journals: Discover world-class research

Abstract

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by pulmonary vasoconstriction, pulmonary arterial remodeling, abnormal angiogenesis and impaired right ventricular function. Despite progress in pharmacological therapy, there is still no cure for PAH. The peptide apelin and the G-protein coupled apelin receptor (APLNR) are expressed in several tissues throughout the organism. Apelin is localized in vascular endothelial cells while the APLNR is localized in both endothelial and smooth muscle cells in vessels and in the heart. Apelin is regulated by hypoxia inducible factor-1α and bone morphogenetic protein receptor-2. Patients with PAH have lower levels of plasma-apelin, and decreased apelin expression in pulmonary endothelial cells. Apelin has therefore been proposed as a potential biomarker for PAH. Furthermore, apelin plays a role in angiogenesis and regulates endothelial and smooth muscle cell apoptosis and proliferation complementary and opposite to vascular endothelial growth factor. In the systemic circulation, apelin modulates endothelial nitric oxide synthase (eNOS) expression, induces eNOS-dependent vasodilatation, counteracts angiotensin-II mediated vasoconstriction, and has positive inotropic and cardioprotective effects. Apelin attenuates vasoconstriction in isolated rat pulmonary arteries, and chronic treatment with apelin attenuates the development of pulmonary hypertension in animal models. The existing literature thus renders APLNR an interesting potential new therapeutic target for PH.

Keywords

apelin aPJ Apelin and the apelin receptor pulmonary hypertension

INTRODUCTION

Pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a severe disease with a median survival of 2.8 years if left untreated.^[1] Over the past two decades, novel drugs with a pulmonary vasodilator action and a possible additional inhibitory effect on vascular cell proliferation have been developed, but even after the introduction of such compounds the chance of survival remains poor, with a 3-year survival less than 60%.^[2]

PAH is characterized by a mean pulmonary arterial pressure (MPAP) above 25 mmHg at rest and an increased pulmonary vascular resistance (PVR) in combination with a normal pulmonary capillary wedge pressure (PCWP).^[3] PAH eventually leads to right ventricular pressure overload and compensatory hypertrophy followed by dilatation and failure of the right ventricle,^[4,5] which is the most common cause of death.^[6] The current therapeutic drugs are primarily pulmonary vasodilators such as endothelin-1 (ET-1) receptor antagonists, prostacycline analogues and phosphodiesterase-5 inhibitors that aim to correct for abnormalities in the secretion of endothelium-derived vasoactive mediators. Nevertheless, no current therapy against PAH is sufficient to cure or stop the disease progression. Consequently, there is a need for new therapies.

Pathophysiological mechanisms of PAH

Multiple genetic, cellular and molecular functions are involved in the pathophysiology of PAH. These have recently been reviewed extensively.^[7] A number of pathophysiological mechanisms involved in PAH are relevant in relation to the subject of this paper. For example, normoxic activation of hypoxia-inducible factor (HIF-1α), normally exerting the physiologic hypoxic vasoconstriction, can occur in cells prior to the spontaneous development of PAH in fawn-hooded rats and is thought to be a possible contributor to the development of PAH.^[8] Furthermore, genetic aspects play a role. One of the most prominent genes involved in PAH is the bone morphogenetic protein receptor 2 (BMPR-2), in which mutations occur in 70% of patients with familial PAH and in 25% of patients with idiopathic PAH.^[7]

Abnormal apoptosis and proliferation of vascular endothelial and smooth muscle cells,^[7,9] is involved in the remodeling process of the pulmonary arteries, development of plexiform lesions, and loss of the microvasculature. Numerous humoral factors, including vascular endothelial growth factor (VEGF), are involved in this response.^[9] Furthermore, the function of the endothelium is altered in PAH, resulting in an imbalance between endothelium-derived vasoconstrictors and proliferative agents such as ET-1 and thromboxane, and vasodilators with antiproliferative effects including nitric oxide (NO) and prostacyclin.^[10] In addition to contributing to the remodeling process, it results in decreased vasorelaxation of the pulmonary vascular bed. Angiotensin-II also induces vasoconstriction and mitogenesis in PAH, while enhanced expression of the angiotensin-II converting enzyme 2 (ACE2) has been found to have a beneficial effect in animal models of pulmonary hypertension.^[11,12]

The right ventricle is subjected to pressure-induced alterations in PAH. Compensatory hypertrophy and fibrosis of the right ventricle develops, followed by decreased systolic function and dilatation.^[4] Among other mechanisms, ischemia and apoptosis are central players in this process,^[4] and have increased the interest to investigate whether drugs directly targeting mechanisms in the right ventricle may improve the course of PAH.

Apelin and the apelin receptor

The peptide apelin and the apelin receptor (APLNR) are present in the heart,^[13,14] the systemic and pulmonary vasculature, and the expression of apelin and APLNR is regulated by HIF-1α^[15] and BMPR-2.^[16] Furthermore, the apelin-APLNR system is involved in normal vascular development^[17] and regulation of apoptosis,^[16] and has been shown to be involved in regulation of NO-dependent vasodilatation^[15,18] and to improve cardiac contractility.^[19] Most studies on apelin have addressed the systemic circulation, but the characteristics revealed by these studies have drawn attention to apelin-APLNR as a potential new target in pulmonary hypertension. Recently, support for a role of apelin and APNLR in pulmonary hypertension has emerged through experiments in animal models of pulmonary hypertension.

The purpose of this paper is to introduce the apelin-APLNR system, to review the associations between PAH pathophysiology and apelin based on existing studies, to touch upon apelin as a potential biomarker for PAH, and to point out the regulating effects of apelin on vascular endothelial and smooth muscle cell proliferation, the vasodilatory effect and positive inotropic effect that renders modulation of APLNR an interesting therapeutic target in PAH.

DISCUSSION

Apelin and APLNR

Structure

APLNR was discovered in 1993 by O'Dowd and colleagues who were looking for subtypes of the vasopressin receptor The APLNR was first named APJ and is a G-protein coupled 7 trans-membrane receptor coded for on chromosome 11 band 12, and shares 54% homology in the trans-membrane region with the AT1 receptor. It does not, however, bind angiotensin-II.^[20] Human APLNR shares 74% homology with the rat APLNR.

In 1998, apelin was isolated by Tatemoto et al.^[21] They showed that the active forms of apelin are peptides cleaved from the C-terminal of the 77 amino acids long preproapelin. Fragments of 36, 17, 13 including the N-terminal pyroglutamated apelin-13 (Pyr¹-apelin-13), exist in vivo and have biological activity, but also synthetic fragments of the 12 amino acids from the C-terminal can activate the receptor.^[22] It has been suggested that Pyr^J-apelin-13 is the final active product of apelin, which is more resistant to enzymatic cleavage.^[23,24] Biological differences between apelin fragments have been shown^[25] (e.g., apelin-13, and −17 binds the receptor with higher potency), whereas it is proposed that apelin-36 dissociates slower from the APLNR.^[26–28]

ACE 2 cleaves a single amino acid from the c-terminal of apelin-13 by hydrolysis^[29] (Fig. 1a). It is not fully elucidated whether this inactivates the protein, since studies investigating apelin-17 with amino-acids deleted from the c-terminal found that such peptides still bound to the receptor.^[30] However, the study suggested that receptor activation was altered, in that internalization did not take place.^[19] So far, ACE 2 is the only known enzyme metabolizing apelin. Nevertheless, it seems that the half-life of apelin is short. A half time less than 8 minutes has been documented in humans.^[27]

Figure 1:

Proposed signaling pathways for apelin and APLNR. The figure summarizes data obtained from both systemic and pulmonary vasculature and both left and right ventriclet. A: the structure of apelin-13 showing the site for ACE-2 mediated hydrolysis. B: known pathways in endothelial cells, C: vascular smooth muscle cells, and D: cardiomytes, blue →: Stimulates activity; red →: Decreases activity. Na²+/Ca²+ E: Na²+/Ca²+ exchanger, Na²+/H+ E: Na²+/Ca²+ exchanger.

In short, the difference between the apelin-peptides of different lengths is not fully determined, and furthermore, the pharmacokinetics of the peptides are largely unknown.

Apelin-APLNR interaction and signaling

There is evidence that the APLNR is coupled to an inhibitory G_i-protein because apelin inhibits forskolin induced 3′5″ cyclic adenosine monophospate (cAMP) production in Chinese hamster ovary (CHO) cells^[21,26,31] and because extracellular acidification in CHO cells can be blocked by pertussis toxin.^[26] Furthermore, apelin also works by stimulating the Na²+/H+ exchanger in CHO cells,^[26] and apelin has been shown to activate extracellular signal-regulated kinase (ERK).^[28,32] Some effects of apelin have been shown to be mediated by the phophoinositol-3-phosphokinase (PI-3K)/AKT activated pathway.^[33] Although Masri et al. reported that apelin could not induce increments of inositol phosphate in CHO cells and concluded that the APLNR did not bind a Gq/G11 protein,^[32] the positive inotropic effects of apelin are dependent on the function of phospholipase C (PLC), protein kinase C (PKC), the Na²⁺/H⁺ and the Na²⁺/Ca²+ exchanger,^[19] and an increase in intracellular Ca²+ transients.^[34] These findings are consistent with coupling of the APLNR to a G_q-protein^[35] (Fig. 1).

A 2004 study found that the APLNR was internalized into the cytoplasm and subsequently, the nucleus, upon apelin-APLNR interaction in CHO cells, and it was observed that only apelin fragments able to induce internalization exerted a biological in vivo response.^[30] However, other G-protein coupled receptors such as the angiotensin-II receptor AT₁ are able to elicit biological responses both through activation of the G-protein, but also to act on intracellular pathways after internalization.^[36] Hence, this coupling between internalization and effect requires further investigation.

To date, apelin has not been proven to bind to other receptors,^[31,37,38] and no subtypes of the APLNR have been found. Also, no other endogenous ligands for the APLNR are known, but mice lacking the APLNR are different, as described below, from mice lacking the apelin gene, which suggests that other endogenous substances influence the activity of APLNR.^[39] For example, the APLNR has been shown to form heterodimers and oppose the effect of the angiotensin-II receptor independently of the presence of apelin.^[40]

Localization of APLNR and apelin

Early studies showed that apelin and APLNR mRNA was expressed in numerous tissues of the rat,^[26,41] including several areas of the brain, adipose tissue, heart and lungs. The expression in the lungs was greater than in other tissues, except for a very high expression of apelin in the mammary gland.^[41] In the cardiovascular system, APLNR immunoreactivity was present in both endothelial and smooth muscle cells in the vasculature, in endocardium and myocardium. Apelin immunoreactivity was present in the myocardium, endocardium, and in endothelial cells of the large conduit vessels. In the lungs, apelin immunoreactivity was demonstrated only in the endothelium of small pulmonary arteries, not in the epithelium or vascular smooth muscle cells.^[14] Apelin immunostaining was localized to vesicle-like structures in the endocardial endothelium and in structures associated to the nuclear surface in endothelial cells.^[13] The APLNR has been demonstrated in nuclear fragments of cells.^[42] On the basis of these observations, it has been suggested that apelin is secreted by endothelial cells^[13] and that the APLNR may affect regulation of gene transcription.^[42]

In human tissue, the relative expression of apelin differs from the rat as the mRNA expression of preproapelin and the apelin receptor in the spleen and central nervous system seems higher than in the lungs.^[43] Apelin was also shown to be present in the endothelium of small and larger pulmonary vessels in human tissue.^[44]

Accordingly, the location of apelin in the pulmonary endothelium with receptors in both endothelium and smooth muscle makes this system interesting in relation to pulmonary vascular disease.

Regulation of apelin and APLNR

Factors regulating apelin and APLNR are summarized in Figure 2.

Figure 2:

Factors influencing expression of apelin. Blue →: Stimulates expression; Red →: Decreases expression.

Hypoxia

Vast evidence supports the assertion that apelin expression is regulated by hypoxia. In mice, an increase in total pulmonary apelin mRNA was reported to take place after 1 week of hypoxia,^[45] and in another study in mice, approximately a doubling of apelin mRNA and an increase of apelin protein of about 25% was found after 5 hours of hypoxia.^[46] In rats, however, total pulmonary apelin content was unchanged after exposure to hypoxia for 2 weeks.^[47] An explanation of this apparent incongruence was demonstrated in cultured pulmonary endothelial cells, where apelin mRNA levels were up-regulated after 8 hours of hypoxia, but fell after further hypoxic exposure.^[46] In a study made on mice by Chandra et al., a similar tendency was shown in right ventricular tissue. Here, apelin and APLNR mRNA was four- and three fold increased after one week of hypoxia, but unchanged after 3 weeks.^[48] An initial up-regulation followed by a decrease is also found after glucose deprivation in cultured cardiomyocytes.^[33] The hypoxia-induced regulation of apelin and the APLNR was also found in cultured human hepatocytes, stellate cells^[49] and adipocytes,^[15,50] where APLNR and apelin were up-regulated within 24 hours of hypoxic exposure. Similarly, secretion of apelin to extracellular medium is increased by short-time hypoxia in cultured cardiac myocytes^[51] and adipocytes.^[50] The regulation induced by hypoxia was shown to be mediated by HIF-1α in adipocytes.^[15,50]

In short, it seems that hypoxia regulates the apelin pathway in various tissues, and that there is a biphasic response of apelin and the APLNR to hypoxia with an initial up-regulation followed by a normalization or decrease. Interestingly, in the context of pulmonary hypertension, HIF-1α is a mediator for the hypoxia induced regulation of apelin and APLNR.

Heart failure—mechanical strain or hypoxia?

Apelin and APLNR are altered in heart failure. In an animal model of ischemic heart failure, total heart apelin protein was up-regulated 6 weeks after induction of myocardial infarction.^[52] A study in Dahl salt-sensitive hypertensive rats showed that apelin and APLNR mRNA expression was unaltered in a phase of compensated left heart hypertrophy, but decreased in manifest heart failure,^[53] and in a model of isoprenaline-induced heart failure, apelin and APLNR were found to be down-regulated.^[54]

In one human study, APLNR mRNA and apelin protein levels were up-regulated in left ventricular tissue, but lower in atrial tissue from patients with heart failure compared to healthy controls.^[55] Other studies addressing plasma levels of apelin in heart failure patients have reported down regulation of the apelin system.^[44,56,57] In line with the proposed biphasic response to hypoxia, it is also possible that apelin and APLNR are regulated in opposed directions during different phases of heart failure. This is supported by a study by Chen et al., who showed that apelin plasma levels were in fact higher in patients with low NYHA class heart failure than in healthy controls, but that the levels declined in more advanced functional classes.^[58] Interestingly, cardiac APLNR mRNA rose in patients after implementation of a left ventricular assist device.^[58] This could point towards a regulatory effect of mechanical strain on the heart or other factors involved in heart failure e.g., humoral mediators. To support this, apelin and APLNR were down-regulated in the right ventricle of monocrotaline rats with pulmonary hypertension^[23] and in mice subjected to aortic banding.^[59] On the other hand, tissue hypoxia may also be present in these types of heart failure, and the hypoxia stimulus attenuated by improving left ventricular function. This topic therefore needs more investigation. Apelin levels were also shown to be decreased in patients with ischemic heart disease,^[60–62] and again both hypoxia and mechanical stress may be involved.

BMPR-2, angiotensin-II, insulin and other regulatory factors

A link between the BMPR-2 and apelin was recently recognized.^[16] It was shown that disrupted BMPR2 signaling mediated through Peroxisome proliferator-activated receptor gamma (PPARy)/β-catenin resulted in decreased apelin expression and increased endothelial cell apoptosis. In addition, the authors showed that apelin secreted from pulmonary endothelial cells inhibited proliferation of pulmonary arterial smooth muscle cells. The authors suggested that apelin is a downstream protein from the BMPR-2 receptor signaling involved in pulmonary vascular homeostasis.

Treatment with angiotensin-II has been shown to up-regulate APLNR expression in cultured hepatocytes.^[49] In models of decompensated heart failure APLNR expression was decreased,^[53,63] and in one study, this was prevented by treatment with an angiotensin-II receptor antagonist.^[53] A β-adrenoceptor agonist failed to do so, even though the two drugs improved the systolic function of the left ventricle equally.^[53] These results are apparently contradictory, but indicate a regulatory relationship between the apelin and angiotensin-II systems.

In adipose tissue, insulin up-regulates the expression of apelin in obese mice and insulin levels are related to plasma apelin levels in obese individuals.^[64] Furthermore, growth hormone and tumor necrosis factor-α are proposed as regulators of apelin in adipose tissue.^[65,66]

Overlap between apelin regulation and factors involved in PAH

Accordingly, apelin and APLNR are regulated by hypoxia, which is an important factor in pulmonary vasoconstriction. In addition, HIF-1α, BMPR-2 and angiotensin-II that have all been linked to the pathophysiology of pulmonary hypertension also regulate apelin/APLNR expression. Furthermore, it is possible that mechanical strain and cardiac overload, that are also features of pulmonary hypertension, affects apelin-APLNR levels. Two studies have shown that serum apelin levels in patients with PAH are lower than in controls,^[44,48] and also a decreased content of apelin in pulmonary arterial endothelial cells in patients with PAH has been demonstrated.^[16] This supports a role of apelin and APLNR in pulmonary hypertension. The regulation by insulin makes it interesting to investigate the role of apelin in obesity-related pulmonary hypertension.

Apelin as a plasma biomarker

The changes in plasma apelin levels in heart failure^[44,55–58] and pulmonary hypertension^[44,48] have brought forward the idea of apelin as a possible biomarker for these diseases. In heart failure patients,^[55] the concentration of apelin was 200 fold higher in atrial tissue than in left ventricular tissue. Plasma apelin concentrations correlated to atrial apelin levels, and it was therefore suggested that atrial apelin might be an important source of apelin in plasma.^[55]

However, studies have compared apelin with brain natriuretic peptide (BNP) and the split product N-terminal BNP (NT-proBNP) as biomarkers in heart failure. It has been established that the main source of circulating BNP and NT-proBNP is the heart,^[67] and in patients with myocardial infarct, apelin-36 was inferior to BNP in order to detect decreased left ventricular systolic function.^[68] Similarly, other studies report that NT-proBNP is superior to apelin in correlation with heart failure severity^[69] and prediction of mortality in acute heart failure.^[70]

In pulmonary hypertension, apelin was proposed as a potential lung-derived biomarker, because of the high expression of apelin in the lungs.^[44] If pulmonary apelin was reflected in plasma, it would be a very interesting biomarker yielding information about the small pulmonary artery endothelium given the presence of apelin here.^[13] Nevertheless, an animal study in chronic hypoxic rats with pulmonary hypertension showed that apelin content in the lungs were not reflected in plasma samples and that plasma apelin levels did not correlate to right ventricular pressure^[47] (Fig. 3). On the other hand plasma apelin levels correlated weakly (r² = 0.33) to changes in right ventricular apelin levels (Fig. 3).

Figure 3:

Original figure adapted from data in Andersen et al.^[47] (a): In chronic hypoxic rats, lung concentrations of apelin were not altered in the same direction as plasma apelin by hypoxia and sildenafil treatment. (b): Right ventricular apelin concentrations correlated weakly with plasma apelin levels; (c): Plasma apelin levels were not correlated to right ventricular systolic pressure. RVSP: Right ventricular systolic pressure. ■ = Normoxic rats; ● = Hypoxic rats, ▲ = Hypoxic rats treated with sildenafil 25 mg/kg/day.

In short, it is not fully established, which organs are most important in determining the plasma levels of apelin, but the widespread distribution of apelin probably reduces the use of the peptide as a marker for functional change in a specific organ. At least apelin does not seem to be a lung-derived biomarker for pulmonary hypertension, and, extrapolating the results from studies in patients with left heart disease, the BNPs may be expected to be more specific than apelin in detecting right ventricular failure secondary to pulmonary hypertension.

Effect of apelin in angiogenesis and vascular cell homeostasis

Angiogenesis

In 1996 the APLNR was found to be present in endothelial cells and precursor cells of the embryonic vasculature.^[17,71] Disrupting the apelin/APJ pathway resulted in reduction or loss of intersegmental vessels in frog embryos,^[72] and the authors also showed that apelin was a mitogen and migration factor for endothelial cells. Mice lacking the apelin gene are viable, and postmortem analyses have shown no major anatomical or histological abnormalities. However, APLNR deficient mice show abnormalities in regulation of blood pressure, and in the cardiovascular development. Furthermore, the offspring of APLNR heterozygous mice did not follow a Mendelian order, showing a significantly smaller number of APLNR^−/− mice than expected.^[39] This suggests that the APLNR plays a more pivotal role than apelin per se, and as mentioned previously, that the APLNR may be activated by other ligands.

Apelin has also been shown to be an angiogenic factor in retinal cells.^[73–77] Lack of apelin has been shown to decrease the hypoxia-induced regeneration of vessels in zebra fish,^[46] and in accordance, a study from 2010 showed that lack of apelin worsens necrosis in a model of hind limb ischemia in mice. Furthermore, apelin administration was able to increase the number of larger vessels and decrease necrosis in a model of hind limb ischemia in mice.^[78]

Apelin and VEGF—similarities and differences

Like vascular endothelial growth factor (VEGF), apelin inhibits apoptosis in endothelial cells.^[16,79] There are conflicting results about the effect of apelin on apoptosis and proliferation in vascular smooth muscle cells. Some studies found that apelin inhibits apoptosis in umbilical vein smooth muscle cells,^[80] and stimulates proliferation in vascular smooth muscle cells from rat thoracic aortas^[81,82] evoked by activation of the PI3-K/AKT pathway.^[80,81,83] However, a study investigating the effects in pulmonary artery smooth muscle cells found that apoptosis was increased by apelin.^[16] Another characteristic of apelin similar to VEGF is a stimulatory effect on tumour and micro-vessel growth in relation to _cancer.^[80,83,84]

In contrast to apelin or APLNR deficient mice, homozygous VEGF knockout mice die mid-gestational due to severe developmental abnormalities of the circulatory system.^[85] This suggests that VEGF is more important in vascular development than apelin. The case seems to be an inter-relationship between apelin and VEGF in that apelin stimulated the expression of VEGF-A in cell culture.^[49] Moreover, VEGF and apelin combined has been shown to more effectively establish new vessels in animal models of limb ischemia;^[78] and, interestingly, apelin counteracted the increased vascular permeability of new vessels induced by VEGF.^[78] Increased vascular leakage may be involved in the pathobiology of PAH,^[7] and apelin has been shown to reduce lipopolysaccharide-induced vascular leakage in rat lung tissue.^[86]

PAH, apelin and VEGF

In PAH patients, endothelial cells have been shown to be abnormal in that proliferation and migration is enhanced, while angiogenesis is less effective in order to generate structured networks,^[87] and other studies also point toward a disordered angiogenesis in PAH.^[88]

The role of VEGF in pulmonary hypertension is ambiguous since the lack of VEGF clearly worsens the severity of pulmonary hypertension development in animal models,^[89] due to apoptosis of normal endothelial cells. This leads to recruitment of poorly differentiated apoptosis-resistant endothelial cells forming plexiform lesions,^[90–92] and furthermore, smooth muscle cells proliferate abnormally.^[16] On the other hand, it seems that excessive VEGF in advanced stages of PAH contributes to the abnormal endothelial cell proliferation and generation of plexiform lesions.^[93] So far apelin seems to be favorable in pulmonary hypertension. The study by Chandra et al. showed more severe pulmonary hypertension and a more pronounced loss of microvessels in the pulmonary vasculature in chronic hypoxic mice lacking the apelin gene.^[48] This, taken together with the attenuating effect of apelin on pulmonary endothelial cell apoptosis,^[16] may suggest that apelin could modulate the angiogenesis in a positive way in PAH. In pulmonary hypertension related to scleroderma and pulmonary fibrosis, a decreased vascular capacitance is of particular importance,^[94,95] and the effects of apelin in these diseases are worthy of further elucidation.

Effects of apelin on vascular tone and blood pressure

Isolated arteries

In isolated systemic artery^[96,24] and vein^[97] preparations, apelin reduces vascular tone by 25%-50%, and vasoconstriction in response to angiotensin-II has been shown to be inhibited by incubation of arteries with apelin.^[98,99] Other studies^[100] confirm the modulatory effect of apelin on angiotension-II induced vasoconstriction, including a study in APLNR deficient mice that had increased pressor response to angiotensin-II.^[38] However, it was observed that apelin exerted vasoconstriction in denuded vena saphena magna preparations.^[37] In keeping with this, apelin phosphorylates myosin light chain kinase dependent on PKC in vascular smooth muscle.^[101] This suggests that the apelin induced vasodilatation is endothelium-dependent, and that apelin has the opposite effect when acting directly on the smooth muscle cells (Fig. 1).

Blood pressure

One of the first proven physiological effects of apelin was the ability to temporarily lower blood pressure after injection in rats,^[18] and several studies have confirmed this effect,^{[24,25,30,38,102]} including studies showing vasodilation in human volunteers and heart failure patients^{[27, 103]} (Table 1). However, a single study found that apelin given intravenously resulted in an increment of blood pressure in conscious rats^[105] (Table 1). Furthermore, apelin injected or overexpressed in the rostral ventrolateral medulla of the central nervous system results in blood pressure increments.^[106,107]

Table 1:

Effects of apelin on blood pressure in the systemic circulation

Reference	Model	Effect	Duration (in minutes)	APLNR ligand	Dose
Lee et al^[18]	Anaesthetized rat	BP 10-13.0 mmHg ↓	3-4	Apelin-13	1-2 ug/300g i.v.
Charles et al^[102]	Conscious sheep	12 mmHg↓ followed by 12 mmHg↑ in MAP	↓:2 ↑: 4 to 15	Apelin-13	10, 100, 1000 ug i.v.
El Messari^[30]	Anaesthetized rat	MAP 13 mmHg ↓	2	Apelin-17	3 nmol/kg i.v.
Ishida et al.^[38]	Anaesthetized rat, wistar / spontaneously hypertensive rats	MAP 5-9 mmHg ↓ Effect larger in SHY than Wistar rats	-	Pyr¹-apelin-13	2.4-10 nm/l i.v.
Lee et al.^[25]	Anaesthetized SHY rat	MAP 60% ↓	6 minutes, but sustained 20% reduced	Apelin-13	15 ug/kg i.v.
		MAP 30% ↓	6	Apelin-12
	Anaesthetized wistar rat	MAP 30% ↓	6	Apelin-13	15 ug/kg
		MAP 15% ↓		Apelin-12	i.v.
Japp et al.^[27]	Forearm blood fow in human volunteers	Blood fow 3 fold ↑	-	Pyr¹-apelin-13 Apelin-36	0.1 −30 nmol/min i.v.
Cheng et al. ^[104]	Conscious rat	MAP 20 mmHg ↓	-	Apelin-12	20-40 umol/kg i.v.
Kagiyama et al^[105]	Conscious rat	MAP 13 mmHg ↑	-	Apelin-12	20-40 umol/kg i.v.

Studies showing blood pressure reducing effect of apelin in the systemic circulation. MAP: mean systemic arterial blood pressure; BP: blood pressure; i.v.: intravenously; ↓: decrease; ↑: increase

The results suggest that the effects of apelin on blood pressure is not straightforward, and that the net result may depend on the apelin fragment, way of administration, conscious state of the experimental animal, and the propensity for compensatory increases in heart rate and cardiac output.

Apelin and the endothelial NO synthase

Inhibition of prostaglandin synthesis has been shown to both reduce^[24] and be without influence^[97] on the effects of apelin, while numerous studies show that the vasodilatory effect of apelin is inhibited by inhibition of endothelial NO synthase (eNOS).^{[16,27,38,96,97]}

It is also evident that apelin stimulates the activity of eNOS.^[38,98,99] Apelin near-normalized the eNOS phosphorylation in in vitro preparations of the aorta^[99] and renal artery^[98] in diabetic mice with endothelial dysfunction. On the contrary, apelin-induced vasodilatation was reduced in hepatic arteries from patients with liver cirrhosis compared to mesenteric arteries from healthy donor patients,^[96] and this was suggested to be due to a decreased function of eNOS in the arteries from liver cirrhotic patients. However, caution should be taken interpreting these data, since two different vessel types are compared. In short, it is controversial whether apelin normalises impaired eNOS function and improves endothelial function in disease states, or if apelin looses its effects in case of endothelial dysfunction because of decreased eNOS function. AKT,^[99] AMP kinase^[48] and kruppel-like factor 2^[48] have been suggested to link stimulation of APLNR to eNOS (Fig. 1).

Effect of apelin on pulmonary arteries and pulmonary pressure in vitro and in vivo

The effect of apelin in pulmonary arteries has been examined in only a few in vitro studies.^[47,108] One group found that only very high concentrations of apelin were able to transiently decrease the vascular tone with maximally 17%. In a Chinese study, apelin decreased tone by maximally 11%, and the vasorelaxation was abolished by removal of the endothelium and treatment with the eNOS blocker L-NAME^[108] in line with the findings in the systemic circulation. Furthermore, the Chinese group found that the relaxation was decreased by 60% in isolated pulmonary arteries from hypoxic animals. Accordingly, it was shown that in isolated pulmonary resistance arteries from normoxic control rats, treatment with apelin inhibited vasoconstriction to endothelin-1,^[47] while this effect was absent in hypoxic rats with pulmonary hypertension. There was no down-regulation of the APLNR in the arteries from hypoxic rats,^[47] suggesting that the impairment was downstream to the apelin receptor in pulmonary hypertension. This favors the hypothesis that vasodilation induced by apelin is decreased in disease states.

In vivo experiments with dogs subjected to acute pulmonary embolism, showed that apelin injected as a single dose of 20 mg/kg slightly reduced mean pulmonary arterial pressure for approximately 2 minutes. However, the ratio of pulmonary to systemic vascular resistance was not changed.^[109] Long-term studies of apelin in animal models of pulmonary hypertension have shown that it has an attenuating effect on the pulmonary pressure.^[16,23,110] It is, though, difficult to say if this is due to direct vasodilatation, to counteraction or down-regulation of constrictive mediators,^[23] to decreased loss of endothelial cells^[16] and microvasculature,^[48] or to inhibition of smooth muscle cell proliferation.^[16]

PAH, angiotensin-II and apelin

Angiotensin-II has been proposed to contribute to the increased pressure and remodeling in PAH,^[12] and an attenuating effect of apelin on angiotensin-II mediated vasoconstriction in isolated pulmonary arteries from normoxic rats has been observed^[47] in accordance with findings in the systemic circulation. In hypoxic rats with pulmonary hypertension, the inhibitory effect was not present, but this does not exclude that long-term apelin treatment could modulate the effect of angiotensin-II. For example, in cardiomyocytes, apelin blocked angiotensin-II-induced activation of the Rho-A kinase pathway,^[100] which plays a pivotal role in pulmonary hypertension;^[7] and, furthermore, apelin has been shown to antagonize angiotensin-II-induced gene transcription.^[40] The significance of apelin and angiotensin-II sharing ACE 2 as a part of their degradation pathway remains enigmatic in relation to pulmonary hypertension. Exogenous activation of ACE 2 with a synthetic activator has been shown to prevent monocrotaline-induced pulmonary hypertension in rats,^[111] because of an increased breakdown of angiotensin-II to the vasodilatory angiotensin(1-7). How this beneficial effect agrees with increased hydrolysis of apelin by ACE 2 is yet unexplored.

Cardiac effects of apelin-APLNR

Effect on cardiac contractility

An important role for apelin in cardiac function was shown by results from Kuba et al. who observed that apelin-deficient mice developed impaired cardiac contractility with age, unlike their wild type littermates.^[59] Apelin has also been proven to be a potent positive inotropic agent. This was shown for the first time by Szokodi et al. using an isolated perfused heart preparation.^[19] This study showed that apelin was able to increase contractility (by 60% of developed tension) independently of angiotensin-II receptors, endothelin-1 receptors or α-and β-adrenoceptors, and independently of eNOS.

Furthermore, they showed that the positive inotropic effect was reduced by the inhibition of PLC and PKC as well as the Na²⁺-H⁺ exchanger and Na²⁺-Ca²⁺ exchanger (Fig. 1). In this study, very low concentrations of apelin-16 (0.01-10 nM) were used, and thus apelin was characterized as one of the most potent positive inotropic agents known. The inotropic effect has been confirmed by other studies,^{[34,52,54,112]} and interestingly, in normal wild type mice, two weeks infusion of apelin resulted in increased cardiac output, but with no evidence of cardiac hypertrophy.^[113]An interesting report from Japp et al showed increased coronary blood flow, decreased blood pressure, increased contractility and increased cardiac output following acute apelin administration in patients with heart failure.^[103] Some groups have found that the increased contractility was associated with an increment of intracellular Ca²+,^[34] while other studies have shown no increases in Ca²+ transients, but a possible higher myofilament sensitivity towards Ca²⁺.^[114]

Cardioprotective effect of apelin

Improved systolic cardiac function^[115] and reduction in infarct size^[115,116] have been observed in isolated rat hearts after ischemic insult and reperfusion. This was associated to decreased apoptosis,^[33,115] and in the majority of studies,^[33,115,117] the PI3/AKT pathway has been found to mediate this effect (Fig. 1). Another cardioprotective role of apelin is the ability to reduce angiotensin-II-induced cardiac fibrosis.^[100]

Right ventricle, apelin and PAH

The findings of improved contractility without concomitant hypertrophy, and cardioprotective abilities are intriguing in the setting of pulmonary hypertension, because the final consequence of the disease is right ventricular hypertrophy, fibrotic remodeling, and failure followed by death. It has been shown that apelin increases contractility in failing right ventricular myocardium from animals with pulmonary hypertension.^[34] A study addressing the effect of apelin in monocrotaline induced pulmonary hypertension found that apelin was able to normalize contractility indices (dP/dt_max and dP/dt_min) of the right ventricle measured in vivo compared to control animals.^[23] However, it is uncertain whether this finding is due to a decreased pulmonary vascular resistance and right ventricular pressure in the apelin-treated rats or is a direct effect on the right ventricular myocardium.

Therapeutic potential of apelin-APLNR in pulmonary hypertension

Apelin—a new modulator of pulmonary vascular resistance?

The acute vasodilatory effect of apelin on pulmonary arteries^[47,108] and pulmonary pressure^[109] is modest (10%-17%), and is attenuated in pulmonary hypertension. However, this does not exclude the possibility that vasodilatation might occur over a longer period with chronic administration. Apelin could potentially modify secretion of endothelial derived vasoactive factors, as shown in cardiomyocytes.^[23] At any rate, current vasodilatory drugs available in the treatment of PAH are not sufficient to control the disease,^[10] and focus in the management of PAH is now directed towards the basic mechanisms leading to proliferation and pulmonary vascular remodeling. The pressure-reducing effect seen in the long-term experimental models of pulmonary hypertension with chronic administration of apelin^[16,23,110] may possibly be attributed to the stabilizing effect of apelin on endothelial cells,^[16] and the prevention of loss of microvasculature.^[48] Apelin may also affect inflammatory processes, although results are so far sparse and somewhat controversial.^{[40,118–121]}

A new positive inotropic agent?

The benefits of the positive inotropic effect of apelin are uncertain. In heart failure caused by a compromised left ventricle, there are still no proven beneficial effects of any positive inotropic agents on survival despite many clinical studies with several drugs.^[122] Actually, worse outcomes have been observed with β-adrenoceptor stimulating drugs and phosphodiesterase 3 inhibitors,^[123] probably because of increments in energy expenditure and an increased propensity for cardiac arrhytmias, due to increased cAMP in the cardiac conductivity cells.^[124] Even so, the search for useful positive inotropic agents is still ongoing, and the key to a successful drug may lie within the mechanism of which contractility is induced.^[122] The known inotropic mechanisms for apelin differs from those of β-adrenoceptor agonists and phosphodiesterase 3 inhibitors, in that apelin has not been shown to increase levels of cAMP and levels of intracellular calcium may be unaffected.

Speaking in favor of apelin as a promising positive inotropic agent in chronic heart failure are the attenuating effects of angiotensin-II-induced cardiac fibrosis^[100] and the fact that it prevents cardiac apoptosis in relation to ischemia.^[33,115,117]

Potential adverse effects and pharmacological challenges

Apelin increases electrical conduction velocity in the heart^[114] and apelin levels are changed in patients with cardiac arrhytmias.^[125–127] Of 10 sheep injected with apelin, 4 developed some degree of atrio-ventricular block or other abnormalities of the electrocardiogram.^[102] Therefore, the effects of apelin on cardiac conductivity and arrhytmias need closer human testing before clinical application.

Furthermore, obvious challenges for apelin as a drug for treatment of pulmonary hypertension are the short half life and the systemic vasodilatory effect, because PAH patients are already susceptible to symptomatic hypotension.^[128] This may be overcome by pharmacological methods, e.g., chemical modification of the peptide and targeted delivery to the lungs by inhalation. Synthetic forms of apelin with the same biological activity as apelin-13 already exist,^[129] and further development of such analogues may make the modulation of APLNR easier in the future, and allow different routes of administration.

CONCLUSIONS

In summary, apelin and APLNR are present in the pulmonary vasculature, are regulated by factors involved the pathogenesis of PAH, and are down-regulated in pulmonary arterial endothelial cells in patients with PAH. It may not, however, be suited as a specific biomarker for PAH.

Apelin and APLNR play a role in regulation of endothelial and smooth muscle cell homeostasis, and have effects similar and opposite to VEGF. Apelin modulates eNOS expression, induces eNOS-dependent vasodilatation in the systemic and pulmonary circulation, counteracts angiotensin-II-induced vasoconstriction, and has positive inotropic and cardioprotective effects (Fig. 4).

Figure 4:

Factors in PAH pathogenesis opposed by apelin, A: In endothelial cells, B: In smooth muscle cells, and C: Right ventricle. Blue →: Stimulates activity/expression. Red →: Decreases activity/expression.

Thus in the laboratory, the peptide has many intriguing characteristics, but so far most studies have investigated effects in the left heart and systemic circulation, and the peptide has been administered to human subjects in only a very few studies. It should, however, be emphasized that the existing studies in experimental models of pulmonary hypertension point toward a beneficial effect of the drug (Table 2). Further research is needed to clarify the pharmacodynamics, pharmacokinetics and safety of apelin in pulmonary hypertension.

Table 2:

Studies on apelin in isolated pulmonary arteries, PAH patients or PAH animal models

Reference	Model	Main results	APLNR ligand	Dose
Goetze et al,^[44]	PAH patients	PAH patients have lower apelin levels than healthy controls	-	-
Dai et al,^[34]	Chronic hypoxic rats	Apelin increases contractility in right ventricular tissue	Apelin-12	10-70 nM
Falcao-Pires et al,^[23]	Monocrotaline rat	Apelin treatment induces: RVSP ↓Cardiac fibrosis ↓RV expression of ET-1 and ANG-II ↓	Pyr-apelin-13	200 ug/kg/day i.p 17 days
Andersen et al,^[47]	Chronic hypoxic rats Isolated arteries	Contraction to ANG-II and ET-1↓In pulmonary arteries from normoxic rats	Apelin-13	CRC 10⁻¹⁰-3×10⁻⁶ M
Mao et al, ^[130]	Chronic hypoxic rats	MPAP ↓ RVSP/LV+ S ratio ↓	Pyr¹-apelin-13	10 nMol/kg/day, S.C., 28 days
Fan et al,^[110]	Chronic hypoxic rats	MPAP ↓ RVSP/LV+ S ratio i in 10 nMol/kg group	Pyr¹-apelin-13	5-10 nMol/kg/day, S.C. 28 days
Petrescu et al,^[86]	LPS induced pulmonary edema	Extravasation ↓	Apelin-13	2 mg/kg i.p.
Feng et al,^[109]	Acute pulmonary embolism in anaesthetized dogs	MPAP ↓ (<5 mmHg, duration 3 minutes)	Apelin-13	10 and 20 ug/kg i.v.
Chandra et al,^[48]	Apelin deficient mice exposed to chronic hypoxia	Apelin-deficient mice: RVSP ↑Muscularized pulmonary arteries ↑Loss of microvessels ↑Plasma apelin in iPAH patients < controls	-	-
Huang _{et al,}^[108]	Chronic hypoxic rats. Isolated pulmonary arteries	Vascular tone 10% ↓ in normoxic rats	Apelin	CRC 0.01-100 nM
Alastalo et al,^[16]	TIE2CrePPARY^fl/fl mice	Apelin reverses mild pulmonary hypertensionDisruption of BMPR-2 decreases apelin expressionPlasma apelin in iPAH patients > controls	Apelin	200 ug/kg i.p. 14 days

Studies on apelins effect in isolated pulmonary arteries, PAH patients and PAH animal models. RVSP: right ventricular systolic pressure; RV: right ventricle; CRC: concentration-response curves; ET-1: endothelin-1; ANG-II: angiotensin-II; MPAP: mean pulmonary artery pressure; RV/LV+S: right ventricular weight/left ventricular weight + septum; LPS: lipopolysaccharide; i.p.: intraperitoneally; S.C.: subcutaneously; ↓ :decrease; ↑ :increase

Footnotes

Source of Support: Nil,

Conflict of Interest: None declared.

References

Rich

Dantzker

Ayres

Bergofsky

Brundage

Detre

. Primary pulmonary hypertension. A national prospective study. Ann Intern Med 1987;107:216–23.

Humbert

Sitbon

Chaouat

Bertocchi

Habib

Gressin

. Survival in patients with idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension in the modern management era. Circulation 2010;122:156–63.

Galiè

Palazzini

Manes

. Pulmonary hypertension and pulmonary arterial hypertension: A clarification is needed. Eur Respir J 2010;36:986–90.

Bogaard

Abe

Noordegraaf

Vonk A

Voelkel

. The right ventricle under pressure: Cellular and molecular mechanisms of right-heart failure in pulmonary hypertension. Chest 2009;135:794–804.

Simon

. Right ventricular adaptation to pressure overload. Curr Opin Crit Care 2010;16:237–43.

D'Alonzo

Barst

Ayres

Bergofsky

Brundage

Detre

. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med 1991;115:343–9.

Morrell

Adnot

Archer

Dupuis

Jones

MacLean

. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54(1 Suppl):S20–31.

Bonnet

Michelakis

Porter

Andrade-Navarro

Thebaud

Bonnet

. An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: Similarities to human pulmonary arterial hypertension. Circulation 2006;113:2630–41.

Yeager

Nguyen

Belchenko

Colvin

Takatsuki

Ivy

. Circulating Fibrocytes Are Increased in Children and Young Adults with Pulmonary Hypertension. Eur Respir J 2011 [In press].

10.

Lourenco

Fontoura

Henriques-Coelho

Leite-Moreira

. Current pathophysiological concepts and management of pulmonary hypertension. Int J Cardiol 2011 [In press].

11.

Bradford

Ely

Raizada

. Targeting the vasoprotective axis of the renin-angiotensin system: A novel strategic approach to pulmonary hypertensive therapy. Curr Hypertens Rep 2010;12:212–9.

12.

Shenoy

Katovich

Raizada

. ACE2, a promising therapeutic target for pulmonary hypertension. Curr Opin Pharmacol 2011;11:150–5.

13.

Kleinz

Davenport

. Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells. Regul Pep. 2004;118:119–25.

14.

Kleinz

Skepper

Davenport

. Immunocytochemical localisation of the apelin receptor, APJ, to human cardiomyocytes, vascular smooth muscle and endothelial cells. Regul Pep. 2005;126:233–40.

15.

Glassford

Yue

Sheikh

Chun

Zarafshar

Chan

. HIF-1 regulates hypoxia- and insulin-induced expression of apelin in adipocytes. Am J Physiol Endocrinol Metab 2007;293:E1590–6.

16.

Alastalo

Perez

Pham

Sawada

Wang

. Disruption of PPARgamma/beta-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival. J Clin Invest 2011 [In press].

17.

Cleaver

Tonissen

Saha

Krieg

. Neovascularization of the Xenopus embryo. Dev Dyn 1997;210:66–77.

18.

Lee

Cheng

Nguyen

Fan

Kariyawasam

Liu

. Characterization of apelin, the ligand for the APJ receptor. J Neurochem 2000;74:34–41.

19.

Szokodi

Tavi

Foldes

Voutilainen-Myllyla

Ilves

Tokola

. Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility. Circ Res 2002;91:434–40.

20.

O'Dowd

Heiber

Chan

Heng

Tsui

Kennedy

. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene 1993;136:355–60.

21.

Tatemoto

Hosoya

Habata

Fujii

Kakegawa

Zou

. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 1998;251:471–6.

22.

Langelaan

Bebbington

Reddy

Rainey

. Structural insight into G-protein coupled receptor binding by apelin. Biochemistry 2009;48:537–48.

23.

Falcao-Pires

Goncalves

Henriques-Coelho

Moreira-Goncalves

Roncon-Albuquerque

Jr. Leite-Moreira

. Apelin decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2009;296:H2007–14.

24.

Maguire

Kleinz

Pitkin

Davenport

. [Pyr1]apelin-13 identified as the predominant apelin isoform in the human heart: Vasoactive mechanisms and inotropic action in disease. Hypertension 2009;54:598–604.

25.

Lee

Saldivia

Nguyen

Cheng

George

O'Dowd

. Modification of the terminal residue of apelin-13 antagonizes its hypotensive action. Endocrinology 2005;146:231–6.

26.

Hosoya

Kawamata

Fukusumi

Fujii

Habata

Hinuma

Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem 2000;275:21061–7.

27.

Japp

Cruden

Amer

Goudie

Johnston

. Vascular effects of apelin in vivo in man. J Am Coll Cardiol 2008;52:908–13.

28.

Masri

Morin

Pedebernade

Knibiehler

Audigier

. The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments. J Biol Chem 2006;281:18317–26.

29.

Vickers

Hales

Kaushik

Dick

Gavin

Tang

. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 2002;277:14838–43.

30.

El Messari

Iturrioz

Fassot

De Mota

Roesch

Llorens-Cortes

. Functional dissociation of apelin receptor signaling and endocytosis: Implications for the effects of apelin on arterial blood pressure. J Neurochem 2004;90:1290–301.

31.

Habata

Fujii

Hosoya

Fukusumi

Kawamata

Hinuma

. Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim Biophys Acta 1999;1452:25–35.

32.

Masri

Lahlou

Mazarguil

Knibiehler

Audigier

. Apelin (65-77) activates extracellular signal-regulated kinases via a PTX-sensitive G protein. Biochem Biophys Res Commun 2002;290:539–45.

33.

Zhang

Tao

. Apelin protects against cardiomyocyte apoptosis induced by glucose deprivation. Chin Med J (Engl) 2009;122:2360–5.

34.

Dai

Ramirez-Correa

Gao

. Apelin increases contractility in failing cardiac muscle. Eur J Pharmacol 2006;553:222–8.

35.

Japp

Newby

. The apelin-APJ system in heart failure: Pathophysiologic relevance and therapeutic potential. Biochem Pharmacol 2008;75:1882–92.

36.

Hunyady

Catt

Clark

Gaborik

. Mechanisms and functions of AT(1) angiotensin receptor internalization. Regul Pep. 2000;91:29–44.

37.

Katugampola

Maguire

Matthewson

Davenport

. [(125) I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man. Br J Pharmacol 2001;132:1255–60.

38.

Ishida

Hashimoto

Nishiwaki

Iguchi

Harada

. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-typ. 1 receptor in blood pressure in vivo. J Biol Chem 2004;279:26274–9.

39.

Charo

Fajardo

Kawana

Kundu

Sheikh

. Endogenous regulation of cardiovascular function by apelin-APJ. Am J Physiol Heart Circ Physiol 2009;297:H1904–13.

40.

Chun

Ali

Kojima

Kundu

Sheikh

Agrawal

. Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis. J Clin Invest 2008;118:3343–54.

41.

Kawamata

Habata

Fukusumi

Hosoya

Fujii

Hinuma

. Molecular properties of apelin: Tissue distribution and receptor binding. Biochim Biophys Acta 2001;1538:162–71.

42.

Lee

Lanca

Cheng

Nguyen

Gobeil

Jr. . Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors. J Biol Chem 2004;279:7901–8.

43.

Medhurst

Jennings

Robbins

Davis

Ellis

Winborn

. Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neurochem 2003;84:1162–72.

44.

Goetze

Rehfeld

Carlsen

Videbaek

Andersen

Boesgaard

. Apelin: A new plasma marker of cardiopulmonary disease. Regul Pep. 2006;133:134–8.

45.

Sheikh

Chun

Glassford

Kundu

Kutschka

Ardigo

. In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure. Am J Physiol Heart Circ Physiol 2008;294:H88–98.

46.

Eyries

Siegfried

Ciumas

Montagne

Agrapart

Lebrin

. Hypoxia-induced apelin expression regulates endothelial cell proliferation and regenerative angiogenesis. Circ Res 2008;103:432–40.

47.

Andersen

Markvardsen

Hilberg

Simonsen

. Pulmonary apelin levels and effects in rats with hypoxic pulmonary hypertension. Respir Med 2009;103:1663–71.

48.

Chandra

Razavi

Kim

Agrawal

Kundu

de Jesus Perez

. Disruption of the apelin-APJ system worsens hypoxia-induced pulmonary hypertension. Arterioscler Thromb Vasc Biol 2011;31:814–20.

49.

Melgar-Lesmes

Pauta

Reichenbach

Casals

Ros

Bataller

. Hypoxia and proinflammatory factors up-regulate apelin receptor expression in human stellate cells and hepatocytes. Gut 2011 [In press].

50.

Geiger

Muendlein

Stark

Saely

Wabitsch

Fraunberger

. Hypoxia induces apelin expression in human adipocytes. Horm Metab Res 2011;43:380–5.

51.

Ronkainen

Hanninen

Leskinen

Ruas

Pereira

. Hypoxia inducible factor regulates the cardiac expression and secretion of apelin. FASEB J 2007;21:1821–30.

52.

Atluri

Morine

Liao

Panlilio

Berry

Hsu

. Ischemic heart failure enhances endogenous myocardial apelin and APJ receptor expression. Cell Mol Biol Lett 2007;12:127–38.

53.

Iwanaga

Kihara

Takenaka

Kita

. Down-regulation of cardiac apelin system in hypertrophied and failing hearts: Possible role of angiotensin II-angiotensin typ. 1 receptor system. J Mol Cell Cardiol 2006;41:798–806.

54.

Jia

Pan

Zhang

Geng

Zhao

Gerns

. Apelin protects myocardial injury induced by isoproterenol in rats. Regul Pep. 2006;133:147–54.

55.

Foldes

Horkay

Szokodi

Vuolteenaho

Ilves

Lindstedt

. Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure. Biochem Biophys Res Commun 2003;308:480–5.

56.

Chong

Gardner

Morton

Ashley

McDonagh

. Plasma concentrations of the novel peptide apelin are decreased in patients with chronic heart failure. Eur J Heart Fail 2006;8:355–60.

57.

Chandrasekaran

Kalra

Donovan

Hooper

Clague

McDonagh

. Myocardial apelin production is reduced in humans with left ventricular systolic dysfunction. J Card Fail 2010;16:556–61.

58.

Chen

Ashley

Deng

Tsalenko

Deng

Tabibiazar

. Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction. Circulation 2003;108:1432–9.

59.

Kuba

Zhang

Imai

Arab

Chen

Maekawa

. Impaired heart contractility in Apelin gene-deficient mice associated with aging and pressure overload. Circ Res 2007;101:e32–e42.

60.

Kadoglou

Lampropoulos

Kapelouzou

Gkontopoulos

Theofilogiannakos

Fotiadis

. Serum levels of apelin and ghrelin in patients with acute coronary syndromes and established coronary artery disease–KOZANI STUDY. Transl Res 2010;155:238–46.

61.

Bai

. Reduced apelin levels in stable angina. Intern Med 2008;47:1951–5.

62.

Weir

Chong

Dalzell

Petrie

Murphy

Steedman

. Plasma apelin concentration is depressed following acute myocardial infarction in man. Eur J Heart Fail 2009;11:551–8.

63.

Fukushima

Kobayashi

Takeshima

Koguchi

Ishimitsu

. Effects of olmesartan on Apelin/APJ and Akt/endothelial nitric oxide synthase pathway in Dahl rats with end-stage heart failure. J Cardiovasc Pharmacol 2010;55:83–8.

64.

Boucher

Masri

Daviaud

Gesta

Guigne

Mazzucotelli

. Apelin, a newly identified adipokine up-regulated by insulin and obesity. Endocrinology 2005;146:1764–71.

65.

Daviaud

Boucher

Gesta

Dray

Guigne

Quilliot

. TNFalpha up-regulates apelin expression in human and mouse adipose tissue. FASEB J 2006;20:1528–30.

66.

Kralisch

Lossner

Bluher

Paschke

Stumvoll

Fasshauer

. Growth hormone induces apelin mRNA expression and secretion in mouse 3T3-L1 adipocytes. Regul Pep. 2007;139:84–9.

67.

Mair

. Biochemistry of B-type natriuretic peptide - where are we now?. Clin Chem Lab Med 2008;46:1507–14.

68.

Tycinska

Sobkowicz

Mroczko

Sawicki

Musial

Dobrzycki

. The value of apelin-36 and brain natriuretic peptide measurements in patients with first ST-elevation myocardial infarction. Clin Chim Acta 2010;411:2014–8.

69.

Miettinen

Magga

Vuolteenaho

Vanninen

Punnonen

Ylitalo

. Utility of plasma apelin and other indices of cardiac dysfunction in the clinical assessment of patients with dilated cardiomyopathy. Regul Pep. 2007;140:178–84.

70.

van Kimmenade

Januzzi

Jr. Ellinor

Sharma

Bakker

Low

. Utility of amino-terminal pro-brain natriuretic peptide, galectin-3, and apelin for the evaluation of patients with acute heart failure. J Am Coll Cardiol 2006;48:1217–24.

71.

Devic

Paquereau

Vernier

Knibiehler

Audigier

. Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis. Mech Dev 1996;59:129–40.

72.

Cox

D'Agostino

Miller

Heimark

Krieg

. Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo. Dev Biol 2006;296:177–89.

73.

Kasai

Shintani

Oda

Kakuda

Hashimoto

Matsuda

. Apelin is a novel angiogenic factor in retinal endothelial cells. Biochem Biophys Res Commun 2004;325:395–400.

74.

Kasai

Shintani

Kato

Matsuda

Gomi

Haba

. Retardation of retinal vascular development in apelin-deficient mice. Arterioscler Thromb Vasc Biol 2008;28:1717–22.

75.

Kasai

Ishimaru

Kinjo

Satooka

Matsumoto

Yoshioka

. Apelin is a crucial factor for hypoxia-induced retinal angiogenesis. Arterioscler Thromb Vasc Biol 2010;30:2182–7.

76.

Saint-Geniez

Masri

Malecaze

Knibiehler

Audigier

. Expression of the murine msr/apj receptor and its ligand apelin is upregulated during formation of the retinal vessels. Mech Dev 2002;110:183–6.

77.

Saint-Geniez

Argence

Knibiehler

Audigier

. The msr/apj gene encoding the apelin receptor is an early and specific marker of the venous phenotype in the retinal vasculature. Gene Expr Patterns 2003;3:467–72.

78.

Kidoya

Naito

Takakura

. Apelin induces enlarged and nonleaky blood vessels for functional recovery from ischemia. Blood 2010;115:3166–74.

79.

Sakao

Tatsumi

. The effects of antiangiogenic compound SU5416 in a rat model of pulmonary arterial hypertension. Respiration 2011;81:253–61.

80.

Cui

Mao

Wang

Zhang

Xie

. Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways. Amino Acids 2010;39:1193–200.

81.

Liu

Qin

Pan

. PI3K/Akt signaling transduction pathway is involved in rat vascular smooth muscle cell proliferation induced by apelin-13. Acta Biochim Biophys Sin (Shanghai) 2010;42:396–402.

82.

Qin

Pan

Feng

Chen

. Apelin-induced vascular smooth muscle cell proliferation: The regulation of cyclin D1. Front Biosci 2008;13:3786–92.

83.

Tang

Xie

Yuan

Luo

Huang

Cui

. Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line mC3T3-E1 via JNK and PI3-K/Akt signaling pathways. Peptides 2007;28:708–18.

84.

Berta

Kenessey

Dobos

Tovari

Klepetko

Jan

. Apelin expression in human non-small cell lung cancer: Role in angiogenesis and prognosis. J Thorac Oncol 2010;5:1120–9.

85.

Carmeliet

Ferreira

Breier

Pollefeyt

Kieckens

Gertsenstein

. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–9.

86.

Petrescu

Gurzu

Iancu

Indrei

Dumitriu

Chelaru

. Apelin effects on lipopolysaccharide-increased pulmonary permeability in rats. Rev Med Chir Soc Med Nat Iasi 2010;114:163–9.

87.

Masri

Comhair

Asosingh

Koo

Vasanji

. Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 2007;293:L548–54.

88.

Tuder

Chacon

Alger

Wang

Taraseviciene-Stewart

Kasahara

. Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: Evidence for a process of disordered angiogenesis. J Pathol 2001;195:367–74.

89.

Taraseviciene-Stewart

Kasahara

Alger

Hirth

Waltenberger

. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 2001;15:427–38.

90.

Sakao

Taraseviciene-Stewart

Lee

Wood

Cool

Voelkel

. Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells. FASEB J 2005;19:1178–80.

91.

Sakao

Taraseviciene-Stewart

Wood

Cool

Voelkel

. Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth. Am J Physiol Lung Cell Mol Physiol 2006;291:L362–8.

92.

Sakao

Taraseviciene-Stewart

Cool

Tada

Kasahara

Kurosu

. VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells. FASEB J 2007;21:3640–52.

93.

Tuder

Cool

Yeager

Taraseviciene-Stewart

Bull

Voelkel

. The pathobiology of pulmonary hypertension. Endothelium. Clin Chest Med 2001;22:405–18.

94.

Farkas

Gauldie

Voelkel

Kolb

. Pulmonary hypertension and idiopathic pulmonary fibrosis: A tale of angiogenesis, apoptosis, and growth factors. Am J Respir Cell Mol Biol 2011;45:1–15.

95.

Manetti

Guiducci

Ibba-Manneschi

Matucci-Cerinic

. Mechanisms in the loss of capillaries in systemic sclerosis: angiogenesis versus vasculogenesis. J Cell Mol Med 2010;14:1241–54.

96.

Salcedo

Garijo

Monge

Fernandez

Garcia-Villalon

Luis A

Turrion

Sanchez V

. Apelin effects in human splanchnic arteries. Role of nitric oxide and prostanoids. Regul Pep. 2007;144:50–5.

97.

Gurzu

Petrescu

Costuleanu

Petrescu

. Interactions between apelin and angiotensin II on rat portal vein. J Renin Angiotensin Aldosterone Syst 2006;7:212–6.

98.

Zhong

Huang

Yung

Lau

Leung

Wong

. The novel peptide apelin regulates intrarenal artery tone in diabetic mice. Regul Pep. 2007;144:109–14.

99.

Zhong

Huang

Yung

Lau

Lin

. Apelin modulates aortic vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic mice. Cardiovasc Res 2007;74:388–95.

100.

Siddiquee

Hampton

Khan

Zadory

Gleaves

Vaughan

. Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production. J Hypertens 2011;29:724–31.

101.

Hashimoto

Kihara

Ishida

Imai

Yoshida

Toya

. Apelin stimulates myosin light chain phosphorylation in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2006;26:1267–72.

102.

Charles

Rademaker

Richards

. Apelin-13 induces a biphasic haemodynamic response and hormonal activation in normal conscious sheep. J Endocrinol 2006;189:701–10.

103.

Japp

Cruden

Barnes

van

Mathews

Adamson

. Acute cardiovascular effects of apelin in humans: Potential role in patients with chronic heart failure. Circulation 2010;121:1818–27.

104.

Cheng

Pang

. Venous dilator effect of apelin, an endogenous peptide ligand for the orphan APJ receptor, in conscious rats. Eur J Pharmacol 2003;470:171–5.

105.

Kagiyama

Fukuhara

Matsumura

Lin

Fujii

Iida

. Central and peripheral cardiovascular actions of apelin in conscious rats. Regul Pep. 2005;125:55–9.

106.

Yao

Modgil

Zhang

Pingili

Singh

O'Rourke

. Pressor effect of apelin-13 in the rostral ventrolateral medulla: Role of NAD(P)H oxidase-derived superoxide. J Pharmacol Exp Ther 2011;336:372–80.

107.

Zhang

Yao

Raizada

O'Rourke

Sun

. Apelin gene transfer into the rostral ventrolateral medulla induces chronic blood pressure elevation in normotensive rats. Circ Res 2009;104:1421–8.

108.

Huang

Fan

Pan

Gao

Mao

. Effect of apelin on vasodilatation of isolated pulmonary arteries in rats is concerned with the nitric oxide pathway. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2011;27:1–5.

109.

Feng

Tan

Gao

Han

. Hemodynamic effect of apelin in a canine model of acute pulmonary thromboembolism. Peptides 2010;31:1772–8.

110.

Fan

Wang

Mao

Hong

Tian

. Protective and therapeutic effect of apelin on chronic hypoxic pulmonary hypertension in rats. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2010;26:9–12.

111.

Ferreira

Shenoy

Yamazato

Sriramula

Francis

Yuan

. Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension. Am J Respir Crit Care Med 2009;179:1048–54.

112.

Berry

Pirolli

Jayasankar

Burdick

Morine

Gardner

. Apelin has in vivo inotropic effects on normal and failing hearts. Circulation 2004;110:II187–93.

113.

Ashley

Powers

Chen

Kundu

Finsterbach

Caffarelli

. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res 2005;65:73–82.

114.

Farkasfalvi

Stagg

Coppen

Siedlecka

Lee

Soppa

. Direct effects of apelin on cardiomyocyte contractility and electrophysiology. Biochem Biophys Res Commun 2007;357:889–95.

115.

Zeng

Zhang

Wang

Tang

. Apelin protects heart against ischemia/reperfusion injury in rat. Peptides 2009;30:1144–52.

116.

Kleinz

Baxter

. Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase. Regul Pep. 2008;146:271–7.

117.

Simpkin

Yellon

Davidson

Lim

Wynne

Smith

. Apelin-13 and apelin-36 exhibit direct cardioprotective activity against ischemia-reperfusion injury. Basic Res Cardiol 2007;102:518–28.

118.

El-Shehaby

El-Khatib

Battah

Roshdy

. Apelin: A potential link between inflammation and cardiovascular disease in end stage renal disease patients. Scand J Clin Lab Invest 2010;70:421–7.

119.

Hashimoto

Kihara

Imai

Yoshida

Shimoyamada

Yasuzaki

. Requirement of apelin-apelin receptor system for oxidative stress-linked atherosclerosis. Am J Pathol 2007;171:1705–12.

120.

Leeper

Tedesco

Kojima

Schultz

Kundu

Ashley

. Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation. Am J Physiol Heart Circ Physiol 2009;296:H1329–35.

121.

Pitkin

Maguire

Kuc

Davenport

. Modulation of the apelin/APJ system in heart failure and atherosclerosis in man. Br J Pharmacol 2010;160:1785–95.

122.

Metra

Bettari

Carubelli

Bugatti

Dei

Del

. Use of inotropic agents in patients with advanced heart failure: lessons from recent trials and hopes for new agents. Drugs 2011;71:515–25.

123.

Packer

Carver

Rodeheffer

Ivanhoe

DiBianco

Zeldis

. Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group. N Engl J Med 1991;325:1468–75.

124.

Burt

Spray

. Inotropic agents modulate gap junctional conductance between cardiac myocytes. Am J Physiol 1988;254:H1206–10.

125.

Ellinor

Low

MacRae

. Reduced apelin levels in lone atrial fibrillation. Eur Heart J 2006;27:222–6.

126.

Falcone

Buzzi

D'Angelo

Schirinzi

Falcone

Rordorf

. Apelin plasma levels predict arrhythmia recurrence in patients with persistent atrial fibrillation. Int J Immunopathol Pharmacol 2010;23:917–25.

127.

Kallergis

Manios

Kanoupakis

Mavrakis

Goudis

Maliaraki

. Effect of sinus rhythm restoration after electrical cardioversion on apelin and brain natriuretic Peptide prohormone levels in patients with persistent atrial fibrillation. Am J Cardiol 2010;105:90–4.

128.

Schannwell

Steiner

Strauer

. Diagnostics in pulmonary hypertension. J Physiol Pharmacol 2007;58 Supp. 5:591–602.

129.

Hamada

Kimura

Ishida

Kohda

Morishita

Ichihara

. Evaluation of novel cyclic analogues of apelin. Int J Mol Med 2008;22:547–52.

130.

Mao

Hong

Fan

Zhang

Guo

. Effect of apelin on hypoxic pulmonary hypertension in rats: Role of the NO pathway. Sheng Li Xue Bao 2009;61:480–4.

Apelin and Pulmonary Hypertension

Abstract

Keywords

INTRODUCTION

Pulmonary hypertension

Pathophysiological mechanisms of PAH

Apelin and the apelin receptor

DISCUSSION

Apelin and APLNR

Structure

Apelin-APLNR interaction and signaling

Localization of APLNR and apelin

Regulation of apelin and APLNR

Hypoxia

Heart failure—mechanical strain or hypoxia?

BMPR-2, angiotensin-II, insulin and other regulatory factors

Overlap between apelin regulation and factors involved in PAH

Apelin as a plasma biomarker

Effect of apelin in angiogenesis and vascular cell homeostasis

Angiogenesis

Apelin and VEGF—similarities and differences

PAH, apelin and VEGF

Effects of apelin on vascular tone and blood pressure

Isolated arteries

Blood pressure

Apelin and the endothelial NO synthase

Effect of apelin on pulmonary arteries and pulmonary pressure in vitro and in vivo

PAH, angiotensin-II and apelin

Cardiac effects of apelin-APLNR

Effect on cardiac contractility

Cardioprotective effect of apelin

Right ventricle, apelin and PAH

Therapeutic potential of apelin-APLNR in pulmonary hypertension

Apelin—a new modulator of pulmonary vascular resistance?

A new positive inotropic agent?

Potential adverse effects and pharmacological challenges

CONCLUSIONS

Footnotes

References