The role of prolactin/vasoinhibins in cardiovascular diseases

Abstract Prolactin (PRL) is a polypeptide hormone that is mainly synthesized and secreted by the lactotroph cells of the pituitary. There are two main isoforms of PRL: 23‐kDa PRL (named full‐length PRL) and vasoinhibins (including 5.6–18 kDa fragments). Both act as circulating hormones and cytokines to stimulate or inhibit vascular formation at different stages and neovascularization, including endothelial cell proliferation and migration, protease production, and apoptosis. However, their effects on vascular function and cardiovascular diseases are different or even contrary. In addition to the structure, secretion regulation, and signal transduction of PRL/vasoinhibins, this review focuses on the pathological mechanism and clinical significance of PRL/vasoinhibins in cardiovascular diseases.

proteolytic cleavage, the concomitant peptide fragment, as another isoform of PRL (5.6-18 kDa, also called vasoinhibins), acquires antiangiogenic properties. 2 Generally, the balance or the interactions between full-length PRL and vasoinhibins regulate vascular functions. 3,4 PRL/vasoinhibins and vascular function have been reviewed and reported, but most of them focus on the impact on vascular and related signal transduction mechanisms. At present, there is no summary of the molecular structure and disease correlation of PRL/vasoinhibins. In the past decade, accumulating evidence showed the roles of PRL in cardiovascular diseases. 4,5 For instance, PRL levels are positively associated with all-cause mortality in cardiovascular diseases 6 ; 16-kDa PRL induces myocardial damage and is involved in the pathogenesis of peripartum cardiomyopathy (PPCM). 7 PRL and its isoforms have vascular regulation functions, yet our appreciation of the effects of such hormones on cardiovascular health is limited.
In this review, we aimed to summarize the structure, secretion regulation, signal transduction of PRL/vasoinhibins, and their pathological mechanism in vascular remodeling. This review will enable researchers to better understand the role of PRL/vasoinhibins in cardiovascular diseases.

| MOLECUL AR FORMS OF PRL
The PRL gene is unique and located on chromosome 6 in the human genome, 8 containing four introns and five exons and an additional noncoding exon 1a. Transcription of the PRL is regulated by two independent promoter regions. The proximal promoter directs pituitary PRL (pPRL) expression, 9 whereas the distal promoter with a 5000-bp upstream of transcription starting site is responsible for extrapituitary PRL (ePRL) mRNA expression. 9 After cleavage of the 28 amino acid signal peptides, the mature protein containing 199 residues is depicted as the 23-kDa PRL monomer. In addition, numerous variants of PRL have been identified, including big PRL (dimer of the monomeric form), big-big PRL (complexes of monomeric form and IgG autoantibodies), and some variants with smaller molecular weight (14,16,. As shown in Figure 1, a number of variants with a molecular weight between 5.6 10 and 18 kDa are defined as a novel family 11 and named vasoinhibins, as these peptides share blood vessel inhibitory properties. Vasoinhibins are derived from proteolytic cleavage of the full-length PRL near or within the long loop that connects the third and fourth α helices, 8,12 so they contain the NH 2 -terminal part of the mature PRL protein instead of the COOH-terminal fragment.

| S ECRE TI ON AND REG UL ATI ON OF PRL
As shown in Figure 2, pPRL is mainly synthesized and secreted by the lactotroph cells of the anterior pituitary, 8,9 and this process is regulated by a number of prolactin-releasing factors (PRFs) and prolactin-inhibiting factors (PIFs) 13 released from the hypothalamus.
The PIFs include dopamine, gonadotropin-combined peptide, and melanocyte stimulating hormone. 14,15 PRFs include thyroid stimulating hormone releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), angiotensin II, and vasoactive peptide. 16 As the most important PIFs, dopamine inhibits the secretion of pPRL via the type 2 dopamine receptor on the surface of lactotroph cells. 17 And pPRL exerts a negative feedback effect on its own secretion by promoting dopamine secretion in the hypothalamous 17 or in an autocrine manner. 1 Of note, PRL is also produced by cells in numerous extrapituitary tissues (ePRL), including endothelial cells (ECs), 18 fibroblasts, 19 and neuronal and immune cells. 20 The regulation of ePRL is dissimilar to F I G U R E 1 Vasoinhibins are produced from proteolytic cleavage of 23-kDa PRL by several endogenous proteolytic enzymes, such as cathepsin D, matrix metalloproteinase (MMP), and bone morphogenetic protein-1 (BMP-1). 35 And vasoinhibins contain the NH 2 -terminal part of the mature PRL protein but not the COOH-terminal fragment. Prolactin (PRL) promotes angiogenesis, whereas vasoinhibins possess antiangiogenic property. The specific impact on vascular is shown in the figure. The solid line has an arrow to indicate the promoting effect, and the other line indicates the inhibiting effect.
that of pPRL and is typically cell-or tissue specific. In decidua, the expression of PRL is controlled by many cytokines (IFNγ and IL-2), transcription factors (Ets-1), and signaling peptides (cAMP and protein kinase A) that act either via well-defined regulatory pathways or by direct binding to putative control elements within the PRL promoter regions. 20 In peripheral blood mononuclear cells, PRL levels can be regulated by calcitriol (the hormonal form of vitamin D). 20 Very little is known about ePRL regulation in the vasculature. Previous study showed that PRL expressed in ECs could act in an autocrine manner to regulate cell proliferation. 21 In addition, STAT5/PRL/vascular endothelial growth factor (VEGF) signaling cascade was proven to exist in human brain ECs and implicate PRL and VEGF as autocrine regulators of EC migration, invasion, and tube formation. 22 Pituitary vasoinhibin generation is closely intertwined with PLR production, as vasoinhibins are produced from proteolytic cleavage of 23-kDa PRL by several endogenous proteolytic enzymes, such as cathepsin D, 12 matrix metalloproteinase (MMP), 23 and bone morphogenetic protein-1. 24 However, the ratio of vasoinhibin generation to PRL synthesis is not fixed; instead, it varies under physiological control. Previous studies in rodents and humans revealed that the ratio of pituitary vasoinhibin to PRL can increase from 0.22 to 0.77 after pregnancy and to 0.99 after perphenazine treatment, a dopamine D1 and D2 receptor antagonist. 11 The ratio is also increased by treatment with estrogen and decreased by TRH. 11 In addition, oxidative stress increases the activity of cathepsin D to cleave PRL, 25 whereas hypoxia decreases cathepsin D-induced vasoinhibin generation. 26 Except for the pituitary, vasoinhibins can be produced in other tissues, including the human endothelium, the placenta, the cartilage, the retina, and the heart. 9 At the local tissue, the vasoinhibin levels are under the regulation of both utilization of circulating and locally produced PRL and the level of activity of local PRL cleaving enzymes, 11 indicating that the microenvironment is important in the regulation of local isoforms of PRL. 11

| PRL RECEP TOR AND S I G NALIZ ATI ON
PRL activities are normally mediated by its specific highly affinitive receptor (PRLR), which is expressed in many tissues, especially the liver, breast, adrenal gland, and hypothalamus. 27 The PRLR is a member of the hematopoietic cytokine receptor superfamily and encoded by a gene located on chromosome 5p13-14. PRLR consists of an extracellular domain that binds PRL, a single transmembrane domain, and a cytoplasmic domain. 28 The main isoform found in humans is a long near-ubiquitous 598 amino acid protein and has a mass of 90 kDa. Due to alternative splicing, there are several different isoforms of PRLR, including short forms, that lack the cytoplasmic domain and predominate in ECs of micro-and macrovascular origins. 29 PRLR can bind at least three ligands, 23-kDa PRL, placental lactogen, and growth hormone, as they are all classical pituitary hormones, structurally, corresponding to a long-chain class-I helical cytokine. 8 When PRL binds to this long isoform of PRLR, several intracellular signaling pathways are activated, including JAK2/STAT (Janus kinase 2/signal transducer and activator of transcription), 8,30 Ras/Raf/ MAPK (mitogen-activated protein kinase), 10,31 and PI3K/Akt (phosphoinositide 3-kinase/protein kinase B) 32 ( Figure 3). Jak2 is a nonreceptor tyrosine kinase that is rapidly active (within 30-60 s) after PRL stimulation, resulting in STAT phosphorylation (STAT1, STAT3, and STAT5) and downstream gene expression, such as VEGF, to induce migration, invasion, and tube formation of ECs. 22 The MAPK pathway is another important cascade activated by PRL and involves the SHC/ GRB2/SOS/RAS/RAF intermediaries upstream of MAPK kinases. 1 F I G U R E 2 pPRL (pituitary prolactin) is mainly synthesized and secreted by the lactotroph cells of the anterior pituitary, and this process is mainly regulated by dopamine and thyroid stimulating hormone releasing hormone (TRH) released in the hypothalamus. pPRL exerts a negative feedback effect on its own secretion by affecting dopamine secretion in the hypothalamous or direct action on lactotroph cells. PRL is also produced by autocrine and paracrine cells in numerous extrapituitary tissues (ePRL), such as immune cells and vascular endothelial cells. 85 Promotion is indicated by a solid line with arrow, and the other line indicates inhibition.
Due to the unique structure, vasoinhibins lose the ability to bind PRLR and cannot activate similar intracellular signaling pathways as the full-length PRL. 1 A specific, high-affinity, saturable binding site was reported on the membranes of capillary ECs decades ago, although its structure has not been identified yet. 33 And then, Khalid Bajou 34 identified plasminogen activator inhibitor-1 (PAI-1) as a binding partner of 16-kDa PRL, and a multicomponent complex formed by PAI-1, urokinase, and the urokinase receptor is required for the full antiangiogenic activity of 16-kDa PRL on ECs. These two binding partners or receptors mediate the vasoinhibin blockage of various signaling pathways, such as Ras-Raf-MAPK, Ras-Tiam1-Pak1, and PLCγ-IP3-eNOS. 11,35

| PRL IN REG UL ATI ON OF VA SCUL AR FUN C TIONS
The vascular actions and signaling mechanisms of PRL and vasoinhibins have been discussed in various reviews. 1,35,36 In summary, circulating or local PRL acts on ECs, immune cells, fibroblasts, pericytes, and smooth muscle cells in a paracrine/autocrine manner, thereby stimulating or inhibiting the proliferation, dilation, permeability, and regression of blood vessels. 5 These opposite effects exist as the fulllength PRL (23 kDa) and promote angiogenesis, 10 but the proteolytic isoforms of PRL, vasoinhibins, acquire antiangiogenic properties. 23,28 In general, ECs (the main vascular intimal cells), smooth muscle cells

| Endothelial cells
ECs line the inner surface of vessels to support tissue growth and repair. Apoptosis by EC injury is usually considered as the initiating factor of vascular remodeling, and its excessive proliferation and apoptosis resistance lead to vascular intimal thickening, lumen stenosis, and even occlusion. PRL was reported to promote cell migration, invasion, and tube formation in ECs through JAK2-STAT5 pathway 22 and to decrease vasopermeability by upregulating the expression of tight-junction proteins between ECs. 37 Meanwhile, PRL stimulates the expression of proangiogenic factors by activating various non-ECs, such as fibroblast growth factor-2 (FGF-2) and VEGF. In addition, heme-oxygenase-1 is confirmed to be the second messenger for PRL-mediated angiogenesis and EC proliferation. 18 Therefore, all the findings suggest that PRL acts not only as a systemic but also as an autocrine/paracrine positive regulator of angiogenesis. 11,35 F I G U R E 3 Signal pathways that may be involved in the combination of PRLR and PRL (prolactin): canonical Janus kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphatidylinositol-3kinase (PI3K)/Akt pathways. Signal transduction pathways are known to be activated in endotheliall cells by vasoinhibins. 39 The binding of vasoinhibins to its receptor and the recognition and localization of vasoinhibins are unknown, including increasing the expression of PAI-1, activating Bcl Xs and/or NFkB, and blocking the stimulating effect of vascular endothelial growth factor (VEGF) on eNOS, RAS-MAPK pathway, or RAS-PAK1 pathway. Promotion is indicated by a solid line with arrow, and the other line indicates inhibition. IP3, inositol trisphosphate; PAI-1, plasminogen activator inhibitor-1; PLC, phospholipase C; u-PA, urokinase-type plasminogen activator.
On the contrary, vasoinhibins inhibit angiogenesis, vasodilation, and vasopermeability by inhibiting the action of several vasoactive substances, such as VEGF, FGF-2, and interleukin 1β (IL-1β), via the Ras-Raf-MAPK pathway and the Ras-Tiam1-Rac1-Pak1 pathway, as well as microRNA-146a 38 on ECs. In addition to these pathways, vasoinhibins may block the mechanism of eNOS activation by interfering with intracellular Ca 2+ calmodulin binding, blocking acetylcholine and bradykinin-induced Ca 2+ transients in ECs. 39 Besides, vasoinhibins can act as a potent pro-inflammatory cytokine that stimulates iNOS expression and NO production, and exogenous NO can reverse the inhibition of vasoinhibins on VEGF-induced EC proliferation. 19 Subsequently, NO stimulates cGMP production and activates cGMP-dependent protein kinase (PKG), thus leading ultimately to the activation of Raf-MAPK signaling. 39 However, the effects of PRL on the angiogenic process may be much more complex.
Although PRL can promote angiogenesis at different anatomical sites in the body, the other mediators in the milieu of specific tissue or organ could affect the pro-angiogetic feature of PRL. For example, PRL-mediated proliferation may not occur when PRLR in the vascular endothelium is already occupied by locally produced hormones. 18 The aforementioned signal pathway is shown in Figure 3.

| Immune cells
Immune cells are an important location for PRL production; they participate in angiogenesis by producing and releasing a large number of proangiogenic mediators. 36 Simultaneously, they also have antian-

| Smooth muscle cells
Smooth muscle cells, a major structural component of the vessel wall, provide the main support for the structure of the vessel wall and regulate vascular tone to maintain intravascular pressure and tissue perfusion. 48 Sauro and Zorn 49 found that PRL-induced aortic smooth muscle cell proliferation is mediated through PKC pathway, suggesting the role of PRL in vascular smooth muscle cell hyperplasia and the pathogenesis of cardiovascular diseases such as hypertension and atherosclerosis. However, the research on PRL affecting vascular function by stimulating smooth muscle cells is insufficient, which needs to be further explored.

| PHYS IOLOG IC AL AND CLINI C AL IMPLI C ATI ON S OF PRL IN C ARDIOVA SCUL AR DIS E A S E S
A large amount of evidence showed that there is a causal relationship between PRL and cardiovascular diseases, 50,51 which is summarized in Table 1. The roles of PRL in cardiovascular disorders are discussed in the following section. RWT, relative wall thickness; SDS, Zung self-rated depression scale; VO 2 max, peak oxygen uptake.

| Arteriosclerosis
Atherosclerosis is a chronic inflammatory disease; unstable atherosclerotic plaque rupture, vascular stenosis, or occlusion caused by platelet aggregation and thrombosis lead to acute cardiovascular diseases. 52 Several studies suggested hyperprolactinemia contributes to the development of atherosclerosis. 53 In menopausal women, PRL, even at normal levels, was found to positively correlate with blood pressure (BP), arterial stiffness, and the Heart Score of the European Society of Cardiology (a composite index that predicts mortality within 10 years). 54 However, inverse associations between PRL and left ventricular mass change, incident left ventricular hypertrophy, and altered left ventricular (LV) geometry were observed in men rather than in women. 55 Besides, patients with hyperprolactinemia have significant increases in mean carotid intima thickness, capillary blood glucose, insulin resistance, and hypersensitive C-reactive protein. 56  In PPCM mice, a cardiomyocyte-specific knockout for STAT3

| Pulmonary hypertension
Pulmonary hypertension (PH) is a lethal and progressive cardiovascular disorder characterized by pulmonary vascular remodeling, resulting in increased pulmonary artery pressure and progressive right ventricular dysfunction. 65 A previous study showed that serum levels of PRL and 16-kDa PRL were significantly higher in patients with precapillary PH, which was negatively correlated with 6-minute-walk test and peak oxygen uptake. 66

| Chronic heart failure
Heart failure encompasses heart dysfunction due to any type of cardiovascular diseases. Serum PRL levels have been shown to have a significantly negative correlation with left ventricular ejection fraction (LVEF), 6-minute-walk test, and natriuretic peptides in patients with chronic heart failure, and patients who had higher baseline PRL levels were at high risk of death or hospitalization. 82 In this study, the author also found that PRL levels were significantly associated with the proinflammatory markers IL-6 and TNFα and the anti-inflammatory cytokine IL-10 and proposed that a vicious combination of PRL, oxidative stress, and inflammation may attribute to the pathogenesis of chronic heart failure. 82 However, whether prolactin elevation is a causal factor of chronic heart failure remains to be elucidated.

| Retinopathy of prematurity
Retinopathy of prematurity (ROP) is a potentially blinding retinal neovascularization disease. In a prospective, case-control study, serum PRL and vasoinhibins were measured weekly in 90 preterm infants diagnosed with ROP or controls between 1 and 9 weeks after birth. PRL levels were found to be higher in ROP patients than in controls during the first (vasoinhibitory) and the second (vasoproliferative) phases of ROP. 83 Although vasoinhibin levels (combined with 14-and 16-kDa PRL) significantly increased during the first week after birth in ROP patients, the levels became equal to those in controls during the postnatal weeks, which indicated that dysregulation of the PRL/vasoinhibin axis Yuxia Huang and Shang Wang drew the figures. All authors contributed to manuscript revision, read, and approved the submitted version.

ACK N OWLED G M ENTS
The authors acknowledge the use of Servier Medical Art image bank that was used to create Figures 1-3.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.