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Objectives. Prolactin (PRL) is a lactation-inducing hormone with immunomodulatory properties and is found at elevated levels in the serum of patients with RA and other rheumatic diseases. The PRL receptor (PRLR) has been shown to be expressed by macrophages in atherosclerotic plaques. The aim of this study was to examine PRLR expression by synovial macrophages and its role in the regulation of macrophage activation. Methods. Serum monomeric 23 kDa PRL levels were measured in 119 RA patients using a fluoroimmunometric assay. PRLR expression was assessed in synovial tissue of 91 RA, 15 PsA and 8 OA patients by immunohistochemistry and digital image analysis. Double IF was used to identify PRLR-expressing cells. The effects of PRL on monocyte-derived macrophage gene expression were examined by quantitative real-time PCR and ELISA. Results. Serum PRL levels were similar in female and male RA patients. Median (interquartile range) PRLR expression was significantly higher (P < 0.05) in RA and PsA synovial tissue compared with OA. PRLR colocalized with synovial CD68(+) macrophages and von Willebrand factor(+) endothelial cells. In vitro, PRLR was prominently expressed in IFN-gamma-and IL-10-polarized macrophages compared with other polarizing conditions. PRL by itself had negligible effects on macrophage gene expression, but cooperated with CD40L and TNF to increase expression of pro-inflammatory genes including IL-6, IL-8 and IL-12 beta. Conclusions. Synovial PRLR expression is enhanced in patients with inflammatory arthritis compared with OA, and PRL cooperates with other pro-inflammatory stimuli to activate macrophages. These results identify PRL and PRLR as potential new therapeutic targets in inflammatory arthritis.

Background and Purpose Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Experimental Approach Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Key Results Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (−31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (−70%) and secretion (−28%) by peritoneal macrophages. Conclusions and Implications Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis.

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.

Atherothrombosis is a multifactorial process, governed by an interaction between the vessel wall, hemodynamic factors and systemic atherothrombotic risk factors. Recent in vitro, human ex vivo and animal studies have implicated the hormone prolactin as an atherothrombotic mediator. To address this issue, we evaluated the anatomy and function of various microvascular beds as well as plasma atherothrombosis markers in patients with elevated prolactin levels. In this pilot study, involving 10 prolactinoma patients and 10 control subjects, sidestream dark field (SDF) imaging revealed a marked perturbation of the sublingual microcirculation in prolactinoma patients compared to control subjects, as attested to by significant changes in microvascular flow index (2.74 +/- A 0.12 vs. 2.91 +/- A 0.05, respectively; P = 0.0006), in heterogeneity index (0.28 [IQR 0.18-0.31] vs. 0.09 [IQR 0.08-0.17], respectively; P = 0.002) and lower proportion of perfused vessels (90 +/- A 4.0% vs. 95 +/- A 3.0%, respectively; P = 0.016). In the retina, fluorescein angiography (FAG) confirmed these data, since prolactinoma patients more often have dilatated perifoveal capillaries. In plasma, prolactinoma patients displayed several pro-atherogenic disturbances, including a higher endogenous thrombin potential and prothrombin levels as well as decreased HDL-cholesterol levels. Prolactinoma patients are characterized by microvascular dysfunction as well as plasma markers indicating a pro-atherothrombotic state. Further studies are required to assess if prolactin is causally involved in atherothrombotic disease.

SummaryPlatelets play an important role in the development of plaque formation and in the events after rupture of the atherosclerotic plaque, leading to atherothrombosis. Multiple hormones, either in excess or when deficient, are involved in the development of atherothrombotic disease, but, to which extent such hormones affect platelet function, is still controversial. It was the objective of this study to assess the ability of the pituitary hormone prolactin to affect platelet functions. Venous blood was collected from six healthy males. Platelet activation was studied by (i) flow cytometry in whole blood (exposure of P-selectin as a measure of platelet secretion, and binding of PAC-1 as a measure of ligand-binding conformation of αIIbβ3), and by (ii) optical aggregation and whole blood aggregation. All studies were performed without and with exposure to several concentrations of ADP (0.1, 0.5 and 1.0 μM) and prolactin (50 and 1,000 μg/l). The presence of the prolactin receptor was investigated by Western blot and flow cytometry. In response to either 50 or 1,000 μg/l prolactin, no evidence of platelet activation or aggregation was found. In addition, ADP-induced platelet activation or aggregation was not enhanced by prolactin. Finally, prolactin receptors could not be detected on the surface of platelets. The present data indicate that prolactin does not directly modulate platelet function.

The pituitary-derived hormone prolactin has been suggested to stimulate the development of atherosclerosis and cardiovascular disease through its effects on metabolism and inflammation. In this study, we aimed to challenge the hypothesis that inhibition of prolactin function may beneficially affect atherosclerosis burden. Hereto, atherosclerosis-susceptible LDL receptor (Ldlr) knockout mice were transplanted with bone marrow from transgenic mice expressing the pure prolactin receptor antagonist Del1-9-G129R-hPRL or their non-transgenic littermates as control. Recipient mice expressing Del1-9-G129R-hPRL exhibited a decrease in plasma cholesterol levels (−29%; PP3 μm2 for Del1-9-G129R-hPRL vs 259±32×103 μm2 for controls) and the lesional macrophage and collagen contents were not different between the two groups of bone marrow recipients. Importantly, Del1-9-G129R-hPRL exposure increased levels of circulating neutrophils (+91%; PPPPLdlr knockout mice. Despite an associated decrease in VLDL/LDL cholesterol levels, application of the prolactin receptor antagonist Del1-9-G129R-hPRL does not alter the susceptibility for initial development of atherosclerotic lesions probably due to the parallel increase in circulating leukocyte concentrations.

Atherosclerotic vascular disease is the consequence of a chronic inflammatory process, and prolactin has been shown to be a component of the inflammatory response. Additionally, recent studies indicate that prolactin contributes to an atherogenic phenotype. We hypothesized that this may be the result of a direct effect of prolactin on atherogenesis through activation of the prolactin receptor. Human carotid atherosclerotic plaques were obtained from patients by endarteriectomies. The mRNA of prolactin receptor, but not of prolactin, was detected in these atherosclerotic plaques by quantitative real-time PCR. In situ hybridization confirmed the expression of the prolactin receptor in mononuclear cells. Analysis at the protein level using immunohistochemistry and immunoelectron microscopy revealed that the prolactin receptor was abundantly present in macrophages near the lipid core and shoulder regions of the plaques. Our findings demonstrate that the prolactin receptor is present in macrophages of the atherosclerotic plaque at sites of most prominent inflammation. We therefore propose that prolactin receptor signaling contributes to the local inflammatory response within the atherosclerotic plaque and thus to atherogenesis. Journal of Endocrinology (2011) 208, 107-117

OBJECTIVE: The aim of this trial was to determine whether obese patients benefit from treatment with rimonabant in terms of progression of carotid atherosclerosis. Rimonabant, a selective cannabinoid-1 receptor blocker, reduces body weight and improves cardiometabolic risk factors in patients who are obese. DESIGN, SETTING, PATIENTS, INTERVENTIONS AND RESULTS: A prospective, double-blind, placebo-controlled trial (Atherosclerosis Underlying Development assessed by Intima-media Thickness in patients On Rimonabant (AUDITOR)) randomised 661 patients with abdominal obesity and metabolic syndrome to rimonabant or placebo for 30 months of treatment. The absolute change in the average value for six segments of far wall carotid intima-media thickness from baseline to month 30 was 0.010 ± 0.095 mm in the rimonabant group and 0.012 ± 0.091 mm in the placebo group (p=0.67). The annualised change was an increase of 0.005 ± 0.042 mm for the rimonabant-treated group and 0.007 ± 0.043 mm for the placebo-treated group (p=0.45). CONCLUSIONS: There was no difference in atherosclerosis progression between patients receiving rimonabant for 30 months and those receiving placebo for the primary efficacy measure (absolute change in carotid intima-media thickness). These findings are consistent with a similar study using coronary intravascular ultrasound and another study evaluating the occurrence of cardiovascular events. Our findings suggest that a 5% loss of body weight over a 30-month period with rimonabant is insufficient to modify atherosclerosis progression in the carotid artery in obese patients with metabolic syndrome. Clinical trial registration information clinicaltrials.gov Identifier: NCT00228176

Objective— Several acquired risk factors for venous thrombosis (VT) are associated with high prolactin levels. Our goal was to investigate VT risk for different levels of prolactin. Methods and Results— We used data of a case-control study on leg vein thrombosis conducted between September 1999 and August 2006 at the Academic Medical Center, Amsterdam, the Netherlands. Prolactin was assessed in 187 cases (mean age, 57 years; range, 19 to 90) and 374 gender-matched controls (mean age, 57 years; range, 18 to 93). Odds ratios and 95% CI for VT risk were estimated based on several cutoff levels derived from prolactin levels in controls. Odds ratios for VT risk clearly increased with higher prolactin levels. For prolactin levels above the 75th percentile (8 μg/L), we found an odds ratio of 1.7 (95% CI 1.0 to 2.7) as compared with levels below the 50th percentile (6 μg/L). This further increased up to an odds ratio of 4.7 (95% CI 1.8 to 11.8) for prolactin levels above the 97.5th percentile (16 μg/L). The risk was most pronounced in premenopausal women. Conclusion— Our data suggest that prolactin levels are associated with VT in a dose-dependent fashion. Future studies are needed to evaluate the causality of this relationship.

Background— Prolactin is increasingly recognized to play a stimulatory role in the inflammatory response. Because inflammation is considered of crucial importance in the development of atherosclerosis, we aimed to evaluate whether prolactin levels are associated with the occurrence of coronary artery disease (CAD). Methods and Results— We performed a nested case-control study in the prospective EPIC-Norfolk cohort. Cases were apparently healthy men and women, aged 45 to 79 years, who developed fatal or nonfatal CAD (n=882). Controls remained free of CAD (n=1490). Overall, systemic prolactin levels did not differ between cases and controls, and people in the highest prolactin tertile did not have a significantly increased risk of developing future CAD (in men, odds ratio, 1.21; 95% CI, 0.92 to 1.61; in women, odds ratio, 1.12; 95% CI, 0.76 to 1.64). However, in a separate immunohistochemical study, the presence of prolactin receptors could be demonstrated in postmortem human coronary artery plaques (preliminary data). Conclusions— Elevated systemic prolactin levels do not predict CAD in the general population. However, prolactin receptors were found in human coronary artery plaques. This observation may indicate a role of prolactin within atherosclerotic plaques. More studies are needed to define the possible role of prolactin in atherosclerotic plaque development.

Type 2 diabetes is characterized by both peripheral insulin resistance and reduced insulin secretion by beta-cells. The reasons for beta-cell dysfunction in this disease are incompletely understood but may include the accumulation of toxic lipids within this cell type. We examined the role of Abca1, a cellular cholesterol transporter, in cholesterol homeostasis and insulin secretion in beta-cells. Mice with specific inactivation of Abca1 in beta-cells had markedly impaired glucose tolerance and defective insulin secretion but normal insulin sensitivity. Islets isolated from these mice showed altered cholesterol homeostasis and impaired insulin secretion in vitro. We found that rosiglitazone, an activator of the peroxisome proliferator-activated receptor-gamma, which upregulates Abca1 in beta-cells, requires beta-cell Abca1 for its beneficial effects on glucose tolerance. These experiments establish a new role for Abca1 in beta-cell cholesterol homeostasis and insulin secretion, and suggest that cholesterol accumulation may contribute to beta-cell dysfunction in type 2 diabetes.