Fragment Of Prolactin Research Articles

Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.

OR17-03 Vasoinhibin Analogs Vi45-51 And Crivi45-51 Lack The Proinflammatory Properties Of Vasoinhibin In Arthritis

Abstract Disclosure: G. Ortiz: None. J.F. García Rodrigo: None. O.F. Martínez-Díaz: None. J.P. Robles: None. M. Zamora: None. X. Ruiz-Herrera: None. E.A. de Los Ríos: None. G. Martinez de la Escalera: None. C. Clapp: None. Vasoinhibin, a fragment of prolactin, has dual actions on inflammatory arthritis. It reduces joint inflammation by the inhibition of pannus vascularization (1) but enhances arthritis by direct proinflammatory effects on synovial fibroblasts (2). Linear (Vi45-51) and cyclic (CRIVi45-51) heptapeptides comprising the 45 to 51 amino acids of vasoinhibin conserve the antiangiogenic effects and potency of whole vasoinhibin, but are easier to produce, stable, and orally active to be developed for clinical use (3). In view of their therapeutic potential in arthritis, we investigated whether Vi45-51 and CRIVi45-51 conserved the proinflammatory effects of vasoinhibin in joint tissues. Synovial fibroblasts (SF) isolated from the knee joint of male C57BL/6 mice were incubated with or without recombinant vasoinhibin of 123 amino acids (positive control), Vi45-51, or CRIVi45-51. As expected, vasoinhibin upregulated the expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) and increased the production of nitric oxide (NO) in SF. However, similar or higher concentrations of Vi45-51 and CRIVi45-51 were inactive. The lack of inflammatory properties suggested improved efficacy of antiangiogenic Vi45-51 and CRIVi45-51 in arthritis. The antigen-induced arthritis (AIA) mouse model was carried out under severe [20 μg of intra-articular administration of methylated serum albumin (mBSA) as antigen] and mild (2.5 μg mBSA) inflammatory conditions to detect the antiangiogenic and proinflammatory effects of vasoinhibin, respectively (2). Consistent with its antiangiogenic properties, intra-peritoneal injection of CRIVi45-51 reduced joint swelling and pannus formation/vascularization in male C57BL/6 mice subjected to severe AIA. Moreover, in agreement with lack of proinflammatory actions, CRIVi45-51 did not elevate joint swelling under mild AIA. We conclude that Vi45-51 and CRIVi45-51 are beneficial in arthritis due to their antiangiogenic properties and lack of proinflammatory actions. Vasoinhibin analogs may be good therapeutic alternatives in inflammatory arthritis, including rheumatoid arthritis. Supported by CONACYT (A1-S-9620B) and UNAM (DGAPA-PAPIIT IN202321). Reference: (1) Ortiz et al., Lab Invest. 2020 Aug;100(8):1068-79. (2) Ortiz et al., Endocrinology 2022 May;163(5): bqac036. (3) Robles et al., Angiogenesis 2022 Feb;25(1):57-70. Presentation: Saturday, June 17, 2023

OR2806 Levosulpiride-Induced Hyperprolactinemia For The Treatment Of Diabetic Macular Edema: A Phase 2 Pilot Clinical Trial

Abstract Disclosure: C. Clapp: None. C.D. Núñez-Amaro: None. M. López: None. E. Adán-Castro: None. M. Robles Osorio: None. R. García-Franco: None. M. García-Roa: None. Y. Villalpando: None. P. Ramírez-Neria: None. N. Pineiro: None. J.F. Rubio-Mijangos: None. J. Sánchez: None. G. Ramirez-Hernandez: None. L. Siqueiros-Márquez: None. N. Díaz-Lezama: None. E. López-Star: None. T. Bertsch: None. G. Martínez de la Escalera: None. J. Triebel: None. Vasoinhibin is a fragment of prolactin that inhibits the permeability and growth of blood vessels. The prokinetic dopamine D2 receptor blocker, levosulpiride, elevates the circulating levels of prolactin and the accumulation of vasoinhibin in the vitreous of patients with proliferative diabetic retinopathy (PDR) (1) suggesting clinical benefits due to the vascular properties of vasoinhibin. Here, we describe the 2-month results of a phase 2 clinical trial (2) investigating the biological activity of levosulpiride in center-involving diabetic macular edema (DME). This prospective, randomized, double-masked, dual-center, placebo-controlled, pilot study was carried out in individuals (aged 40 to 69 years) with DME involving the center of the macula and best-corrected visual acuity (BCVA) between 58 and 16 ETDRS letters at 4 m (20/16 to 20/100 Snellen equivalent) or with PDR undergoing elective pars plana vitrectomy. Oral treatment (TID) with levosulpiride or placebo was for 8 weeks in DME patients or for the 1 week before vitrectomy in PDR patients. Primary outcome measures included changes from baseline in BCVA, central foveal thickness (CFT), and mean macular volume (MMV) every 2 weeks for 8 weeks in placebo- (17 patients, 18 eyes) and levosulpiride- (17 patients, 22 eyes) treated DME groups; whereas vitreous levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) were primary outcomes in PDR patients after placebo (18 patients) or levosulpiride (18 patients). Starting at 4 weeks, levosulpiride improved changes from baseline in BCVA (P &amp;lt; 0.037), CFT (P &amp;lt; 0.013), and MMV (P &amp;lt; 0.002) compared to placebo. At 8 weeks, the proportion of eyes gaining &amp;gt;5 letters (41% vs. 28%), loosing &amp;gt; 21 μm in CFT (55% vs. 28%), and dropping &amp;gt; 0.06 mm3 in MMV (65% vs. 29%) were higher in levosulpiride vs placebo. The overall grading of visual (BCVA) and structural parameters (CFT, MMV, OCT macula image, fundoscopy, and fluorescein angiography) showed improvement with levosulpiride (P = 0.029). Levosulpiride reduced VEGF (P = 0.025) and PlGF (P= 0.008) levels in the vitreous of PDR patients. There were no apparent adverse side-effects. In conclusion, oral levosulpiride administration for 8 weeks improved visual and structural outcomes in patients with center involving DME by mechanisms that may include intraocular upregulation of vasoinhibin and downregulation of VEGF and PlGF. Larger clinical trials evaluating long-term efficacy and safety are warranted. Reference: (1) Núñez-Amaro et al., Transl Vis Sci Technol. 2020 Aug 17;9(9):27. (2) www.clinicaltrials.gov, ID: NCT03161652; Supported by UNAM grant 405PC. Presentation: Sunday, June 18, 2023

The significance of prolactin in systemic connective tissue diseases.

Does prolactin (PRL) level testing in the diagnosis of systemic connective tissue diseases make sense and should we test it in everyday practice? Connective tissue diseases (CTDs) are a group of heterogeneous disorders, involving multiple body systems. Rheumatoid arthritis (RA) is one of the most common connective tissue diseases with a global prevalence of 0.3-1% and can be inherited. Less common are systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), systemic sclerosis (SSc), primary Sjögren's syndrome (pSS) and inflammatory myositis. Prolactin is responsible for lactation, breast growth and many other bodily processes, and is elevated in blood of woman who are pregnant or breastfeeding. Hyperprolactinemia is relatively common in women, so some rheumatic diseases may be caused by high prolactin levels, and it should be detected during diagnosis. Prolactin signals are found in arthritic joint tissues (chondrocytes and synovial fibroblasts) to inhibit cartilage degradation, synovitis and osteoclastogenesis. On the other hand, hyperprolactinemia also promotes the conversion of PRL to vasoinhibin, a fragment of PRL that directly stimulates and indirectly inhibits arthritis in a cell type-dependent manner. The role of the PRL/vasoinhibin axis in inflammatory arthritis should still be monitored and further research is needed to help elucidate the role of PRL in rheumatic diseases in order to ultimately develop new therapeutic interventions that can be tested in patients. Nowadays, there are no clear indications for prolactin testing in the diagnosis of systemic connective tissue diseases. In the case of suspicion or confirmation of some systemic connective tissue diseases, such as rheumatoid arthritis or systemic lupus erythematosus, testing the prolactin level makes sense when severity of disease symptoms is observed.

Anti-Diabetic Therapy and Heart Failure: Recent Advances in Clinical Evidence and Molecular Mechanism.

Diabetic patients have a two- to four-fold increase in the risk of heart failure (HF), and the co-existence of diabetes and HF is associated with poor prognosis. In randomized clinical trials (RCTs), compelling evidence has demonstrated the beneficial effects of sodium-glucose co-transporter-2 inhibitors on HF. The mechanism includes increased glucosuria, restored tubular glomerular feedback with attenuated renin-angiotensin II-aldosterone activation, improved energy utilization, decreased sympathetic tone, improved mitochondria calcium homeostasis, enhanced autophagy, and reduced cardiac inflammation, oxidative stress, and fibrosis. The RCTs demonstrated a neutral effect of the glucagon-like peptide receptor agonist on HF despite its weight-reducing effect, probably due to it possibly increasing the heart rate via increasing cyclic adenosine monophosphate (cAMP). Observational studies supported the markedly beneficial effects of bariatric and metabolic surgery on HF despite no current supporting evidence from RCTs. Bromocriptine can be used to treat peripartum cardiomyopathy by reducing the harmful cleaved prolactin fragments during late pregnancy. Preclinical studies suggest the possible beneficial effect of imeglimin on HF through improving mitochondrial function, but further clinical evidence is needed. Although abundant preclinical and observational studies support the beneficial effects of metformin on HF, there is limited evidence from RCTs. Thiazolidinediones increase the risk of hospitalized HF through increasing renal tubular sodium reabsorption mediated via both the genomic and non-genomic action of PPARγ. RCTs suggest that dipeptidyl peptidase-4 inhibitors, including saxagliptin and possibly alogliptin, may increase the risk of hospitalized HF, probably owing to increased circulating vasoactive peptides, which impair endothelial function, activate sympathetic tones, and cause cardiac remodeling. Observational studies and RCTs have demonstrated the neutral effects of insulin, sulfonylureas, an alpha-glucosidase inhibitor, and lifestyle interventions on HF in diabetic patients.

Cabergoline treatment promotes myocardial recovery in peripartum cardiomyopathy.

Peripartum cardiomyopathy (PPCM) is a rare heart disease, occurring in previously heart-healthy women during the last month of pregnancy or the first months after delivery due to left ventricular (LV) systolic dysfunction. A common pathomechanistic pathway of PPCM includes increased oxidative stress and the subsequent generation of a cleaved prolactin fragment (16kDa PRL), which promotes the onset of heart failure (HF) in a microRNA (miR)-146a-dependent manner. Inhibition of prolactin secretion with the dopamine D2 receptor (D2R) agonist bromocriptine combined with standard HF therapy supports cardiac recovery. This study examined whether treatment with the more selective D2R agonist cabergoline prevents HF development in an experimental PPCM mouse model and might be used as an alternative treatment regime for PPCM. Postpartum (PP) female PPCM-prone mice with a cardiomyocyte restricted STAT3-deficiency (αMHC-Cretg/+ ; Stat3fl/fl ; CKO) were treated over two consecutive nursing periods with cabergoline (CKO Cab, 0.5mg/kg/day) and were compared with bromocriptine treated CKO (CKO Br) and postpartum-matched WT and CKO mice. Cabergoline treatment in CKO PP mice preserved cardiac function [fractional shortening (FS): CKO Cab: 34.5±9.4% vs. CKO: 22.1±9%, P<0.05] and prevented the development of cardiac hypertrophy, fibrosis, and inflammation as effective as bromocriptine therapy (FS: CKO Br: 33.4±5.6%). The myocardial up-regulation of the PPCM biomarkers plasminogen inhibitor activator 1 (PAI-1) and miR-146a were prevented by both cabergoline and bromocriptine therapy. A small cohort of three PPCM patients from the German PPCM Registry was treated with cabergoline (1mg per week for 2weeks, followed by 0.5mg per week for another 6weeks) due to a temporary unavailability of bromocriptine. All PPCM patients initially presented with a severely reduced LV ejection fraction (LVEF: 26±2%). However, at 6months of follow-up, LV function (LVEF: 56±2%) fully recovered in all three PPCM patients, and no adverse events were detected. In the experimental PPCM mouse model, the selective D2R agonist cabergoline prevents the onset of postpartum HF similar to bromocriptine. In PPCM patients, cabergoline treatment was safe and effective as all patients fully recovered. Cabergoline might serve as a promising alternative to bromocriptine. However, these findings are based on experimental data and a small case series and thus have to be interpreted with caution and should be validated in a larger clinical trial.

Case report: the management for a gestational hypertensive woman with influenza A virus pneumonia and peripartum cardiomyopathy

BackgroundPeripartum cardiomyopathy (PPCM) is defined as an idiopathic cardiomyopathy occurring in the last month of pregnancy or the first 6 months postpartum without an identifiable cause. PPCM is suspected to be triggered by the generation of a cardiotoxic fragment of prolactin and the secretion of a potent antiangiogenic protein from the placental, but no single factor has been identified or defined as the underlying cause of the disease. Influenza virus can cause PPCM through immune-mediated response induced by proinflammatory cytokines from host immunity and endothelial cell dysfunction. We report a case in a parturient woman undergoing a cesarean delivery, who had influenza A pneumonia and PPCM.Case presentationA parturient woman at 40 weeks and 1 day of gestation who had experienced gestational hypertension accompanied by pulmonary edema developed hypotension after undergoing an emergency cesarean delivery. An elevation of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) was noted, and echocardiography revealed a left ventricular ejection fraction of 20%. She underwent a nasopharyngeal swab test, in which influenza A antigen was positive. She was diagnosed as having PPCM and received anti-viral treatment. After antiviral treatment, hemodynamic dysfunction stabilized. We present and discuss the details of this event.ConclusionPPCM is a heart disease that is often overlooked by medical personnel. Rapid swab tests, serum creatine kinase measurement, and echocardiography are imperative diagnostic approaches for the timely recognition of virus-associated cardiomyopathy in peripartum women with influenza-like disease and worsening dyspnea, especially during the epidemic season. Prompt antiviral treatment should be considered, particularly after PPCM is diagnosed.

Dual Roles of Prolactin and Vasoinhibin in Inflammatory Arthritis.

The term inflammatory arthritis defines a family of diseases, including rheumatoid arthritis (RA), caused by an overactive immune system, and influenced by host aspects including sex, reproductive state, and stress. Prolactin (PRL) is a sexually dimorphic, reproductive, stress-related hormone long-linked to RA under the general assumption that it aggravates the disease. However, this conclusion remains controversial since PRL has both negative and positive outcomes in RA that may depend on the hormone circulating levels, synthesis by joint tissues, and complex interactions at the inflammatory milieu. The inflamed joint is rich in matrix metalloproteases that cleave PRL to vasoinhibin, a PRL fragment with proinflammatory effects and the ability to inhibit the hyperpermeability and growth of blood vessels. This review addresses this field with the idea that explanatory mechanisms lie within the PRL/vasoinhibin axis, an integrative framework influencing not only the levels of systemic and local PRL, but also the proteolytic conversion of PRL to vasoinhibin, as vasoinhibin itself has dual actions on joint inflammation. In this review, we discuss recent findings from mouse models suggesting the upregulation of endogenous vasoinhibin by the pro-inflammatory environment and showing dichotomous actions and signaling mechanisms of PRL and vasoinhibin on joint inflammation that are cell-specific and context-dependent. We hypothesize that these opposing actions work together to balance the inflammatory response and provide new insights for understanding the pathophysiology of RA and the development of new treatments.

Inhibition of cardiac PERK signaling promotes peripartum cardiac dysfunction

Peripartum cardiomyopathy (PPCM) is a life-threatening heart failure occurring in the peripartum period. Although mal-angiogenesis, induced by the 16-kDa N-terminal prolactin fragment (16 K PRL), is involved in the pathogenesis, the effect of full-length prolactin (23 K PRL) is poorly understood. We transfected neonate rat cardiomyocytes with plasmids containing 23 K PRL or 16 K PRL in vitro and found that 23 K PRL, but not 16 K PRL, upregulated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. Moreover, in bromocriptine-treated CM-KO mice, cardiac function did not improve and cardiomyocyte apoptosis was not suppressed during the peripartum period. These results demonstrate that interaction between 23 K PRL and PERK signaling is cardioprotective during the peripartum term.

Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide

The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.

Vasoinhibin reduces joint inflammation, bone loss, and the angiogenesis and vasopermeability of the pannus in murine antigen-induced arthritis

AbstractIncreased permeability and growth (angiogenesis) of blood vessels play a key role in joint swelling and pannus formation in inflammatory arthritis, a family of diseases influenced by reproductive hormones. The hormone prolactin (PRL) protects against joint inflammation, pannus formation, and bone destruction in adjuvant-induced arthritis and these effects may involve its proteolytic conversion to vasoinhibin, a PRL fragment that inhibits angiogenesis and vasopermeability. Here, we show that the intra-articular injection of an adeno-associated virus type-2 (AAV2) vector encoding vasoinhibin reduced joint inflammation, the hyperplasia, vascular density, and vasopermeability of the pannus, and the loss of bone in mice subjected to antigen-induced arthritis. In agreement, the AAV2 vasoinhibin vector reduced the expression of proinflammatory cytokines (interleukin-1β, interleukin-6), an endothelial cell marker (platelet endothelial cell-adhesion molecule 1), and proangiogenic molecules [vascular endothelial growth factor (VEGF), VEGF receptor 2, and hypoxia-inducible factor 1α] in the arthritic joint. Also, vasoinhibin reduced the synovial vasopermeability induced by the intra-articular injection of VEGF in healthy mice. Finally, vasoinhibin signals by blocking the phosphorylation/activation of endothelial nitric oxide synthase (eNOS) at Ser1179 and the AAV2 vasoinhibin vector inhibited the enhanced phosphorylation of eNOS Ser1179 in the arthritic joint. We conclude that vasoinhibin reduces joint inflammation and bone loss in arthritis by inhibiting pannus angiogenesis and vasopermeability via the blockage of VEGF-induced eNOS activation. These findings suggest the potential therapeutic benefit of AAV2-mediated vasoinhibin gene delivery in arthritis.Adeno-associated virus type 2 encoding the prolactin fragment vasoinhibin (AAV2 vasoinhibin) reduces joint inflammation and bone loss in antigen-induced arthritis in mice by inhibiting pannus angiogenesis and vasopermeability via the blockage of VEGF-induced eNOS activation. These findings suggest the potential benefit of vasoinhibin gene therapy in arthritis.

Prolactin stimulates the vascularisation of the retina in newborn mice under hyperoxia conditions.

The hormone prolactin (PRL) is emerging as an important regulator of ocular blood vessels. PRL is pro-angiogenic and acquires anti-angiogenic properties after undergoing proteolytic cleavage to the PRL fragment, vasoinhibin. The vascularisation of the rodent retina develops after birth when it rapidly expands until completion at the end of the first postnatal week. Exposure of newborn mice to high oxygen levels lowers the rate of blood vessel growth. In the present study, we investigated whether PRL treatment modifies the vascularisation of the retina in newborn mice exposed to high oxygen or to normoxia and whether the retinal conversion of PRL to vasoinhibin may be altered in the neonate. Newborn mice and their nursing mothers were subjected to 75% oxygen or to normoxia from postnatal day (P) 6 to P8 (group 1) or from P2 to P5 (group 2). PRL (2µgg-1 , i.p., twice a day) or vehicle was injected from P5 to P8 in group 1 and from P1 to P5 in group 2. PRL treatment reduced the retinal inhibition of blood vessel growth and the increase in vascular regression induced by hyperoxia as revealed by immunofluorescence staining of blood vessels and the expression of angiogenesis and apoptosis markers. The pro-angiogenic effect may involve a reduced conversion of PRL to vasoinhibin. Incubation of PRL with retinal extracts showed reduced activity of the PRL-cleaving protease, cathepsin D, in the neonate vs the adult retina that was further reduced under hyperoxia. PRL and the PRL receptor mRNA were expressed at higher levels in the retina at P8 than in the adult, whereas endogenous PRL was undetectable in the circulation at P8. We conclude that PRL has a pro-angiogenic effect in the neonate retina as a result of its reduced conversion to vasoinhibin and that PRL produced by the retina may help promote physiological vascularisation after birth.

MON-486 Prolactin Reduces the Hyperoxia-Induced Inhibition of Retinal Neovascularization in Newborn Mice

Retinopathy of prematurity (ROP) is a potentially blinding retinal neovascular disease. Prolactin (PRL) circulating levels are higher in ROP than in control patients and such imbalance is a predictive risk variable for ROP that may impact disease progression (Zepeda-Romero et al. Pediatric Res 2017). PRL accesses the retina from the systemic circulation and is pro-angiogenic but can be cleaved into anti-angiogenic PRL fragments (vasoinhibin). Like preterm infants, newborn mice have incomplete retinal vascularization at birth and exposure to high oxygen, mimicking supplemental oxygen given to premature infants for respiratory care, induces the loss of new blood vessels eventually leading to hypoxia-induced retinal neovascularization. Here, we have investigated whether raising circulating PRL levels modifies the neovascularization of the retina of newborn mice exposed to high-oxygen. Neovascularization was assessed in mice throughout the first 8 days after birth in flat-mounted retinas immunostained for blood vessels. At postnatal day 1 (P1) the retina is almost devoid of blood vessels, which originate from the optic nerve and migrate radially reaching the periphery at P8. Exposure of P6 mice and nursing mothers to hyperoxia (75% oxygen) inhibited retinal vessel growth as determined by a significant (p<0.01) 50% reduction in retinal vascular density and expression of the endothelial cell markers, CD31 and VEGF receptor 2. Treatment with PRL (2 µg/g i.p. twice a day from P5 to P8) elevated PRL levels in serum (>300 ng/ml) and reduced hyperoxia-induced inhibition of retinal neovascularization, as revealed by a significant increase (p<0.03) in retinal vascular density and expression of endothelial cell markers. Current studies are addressing whether the pro-angiogenic effect of PRL is favored by its lower retinal conversion to vasoinhibin under hyperoxic conditions. These findings indicate that high levels of systemic PRL increase the vascularization of the neonatal mouse retina under conditions mimicking an early phase of ROP and support clinical studies suggesting the impact of PRL on disease progression. Supported by CONACYT grant 289568.

Prolactin gene polymorphism in indigenous and crossbred cattle of north east India

Bovine PRL gene is considered one of the important hereditary components of milk productivity, thus seems to be an excellent candidate gene for milk production traits. The study was conducted to study the distribution pattern of allelic variants at the prolactin-RsaI locus in 210 indigenous cattle of north east India (viz. Manipur, Mizoram, Nagaland, Tripura, Asom and Meghalaya,) and crossbred cattle of Mizoram. PCR-RFLP genotyping of a 156 bp fragment of prolactin (PRL) in exon 3 revealed two different allelic variants. The predominant genotype (s) were AB (0.40) and BB (0.40) in indigenous cattle of Manipur, BB in Mizoram (0.57), AB in indigenous cattle of Nagaland (0.50) and Meghalaya (0.63). Whereas in Tripura and Asom indigenous cattle, AA was the most common genotype (0.40 and 0.60). However, AB (0.80) was markedly higher in crossbred (HF × indigenous) cattle. All the six indigenous cattle populations conforming to equilibrium indicated lack of selection pressure for PRL gene in these cattle population. The most frequent allele was A allele in the indigenous cattle of Nagaland (0.55), Tripura (0.55) and Asom (0.70) and B allele in indigenous cattle of Manipur (0.60) and Mizoram (0.73). Whereas, in Meghalaya indigenous and crossbred cattle, both alleles were present in equal frequencies. The findings of differences in the distributions of allelic and genotype variants of PRL-RsaI locus among the indigenous cattle populations of six states of north east India, suggested the presence of scope for genetic selection for milk production traits.

Basis and Design of a Randomized Clinical Trial to Evaluate the Effect of Levosulpiride on Retinal Alterations in Patients With Diabetic Retinopathy and Diabetic Macular Edema.

Diabetic retinopathy (DR) and diabetic macular edema (DME) are potentially blinding, microvascular retinal diseases in people with diabetes mellitus. Preclinical studies support a protective role of the hormone prolactin (PRL) due to its ocular incorporation and conversion to vasoinhibins, a family of PRL fragments that inhibit ischemia-induced retinal angiogenesis and diabetes-derived retinal vasopermeability. Here, we describe the protocol of an ongoing clinical trial investigating a new therapy for DR and DME based on elevating the circulating levels of PRL with the prokinetic, dopamine D2 receptor blocker, levosulpiride. It is a prospective, randomized, double-blind, placebo-controlled trial enrolling male and female patients with type 2 diabetes having DME, non-proliferative DR (NPDR), proliferative DR (PDR) requiring vitrectomy, and DME plus standard intravitreal therapy with the antiangiogenic agent, ranibizumab. Patients are randomized to receive placebo (lactose pill, orally TID) or levosulpiride (75 mg/day orally TID) for 8 weeks (DME and NPDR), 1 week (the period before vitrectomy in PDR), or 12 weeks (DME plus ranibizumab). In all cases the study medication is taken on top of standard therapy for diabetes, blood pressure control, or other medical conditions. Primary endpoints in groups 1 and 2 (DME: placebo and levosulpiride), groups 3 and 4 (NPDR: placebo and levosulpiride), and groups 7 and 8 (DME plus ranibizumab: placebo and levosulpiride) are changes from baseline in visual acuity, retinal thickness assessed by optical coherence tomography, and retinal microvascular abnormalities evaluated by fundus biomicroscopy and fluorescein angiography. Changes in serum PRL levels and of PRL and vasoinhibins levels in the vitreous between groups 5 and 6 (PDR undergoing vitrectomy: placebo and levosulpiride) serve as proof of principle that PRL enters the eye to counteract disease progression. Secondary endpoints are changes during the follow-up of health and metabolic parameters (blood pressure, glycated hemoglobin, and serum levels of glucose and creatinine). A total of 120 patients are being recruited. This trial will provide important knowledge on the potential benefits and safety of elevating circulating and intraocular PRL levels with levosulpiride in patients with DR and DME. Ethics approval has been obtained from the Ethics Committees of the National University of Mexico (UNAM) and the Instituto Mexicano de Oftalmología, I.A.P. Dissemination will include submission to peer-reviewed scientific journals and presentation at congresses. Registered at www.ClinicalTrials.gov, ID: NCT03161652 on May 18, 2017.

Cardiogenic shock complicating peripartum cardiomyopathy: Importance of early left ventricular unloading and bromocriptine therapy.

Acute peripartum cardiomyopathy complicated by cardiogenic shock is a rare but life-threatening disease. A prolactin fragment is considered causal for the pathogenesis of peripartum cardiomyopathy. This analysis sought to investigate the role of early percutaneous mechanical circulatory support with micro-axial flow-pumps and/or veno-arterial extracorporeal membrane oxygenation in combination with the prolactin inhibitor bromocriptine in refractory cardiogenic shock complicating peripartum cardiomyopathy. In this single-centre analysis, five peripartum cardiomyopathy patients with refractory cardiogenic shock received mechanical circulatory support with either Impella CP microaxial pump only (n=2) or in combination with veno-arterial extracorporeal membrane oxygenation (n=3) in the setting of biventricular failure. All patients were mechanically ventilated. In all cases mechanical circulatory support was combined with bromocriptine therapy and early administration of levosimendan. All patients survived the acute phase of refractory cardiogenic shock. Mechanical circulatory support using a micro-axial pump allowed to significantly reduce catecholamine dosage. Remarkably, early left ventricular support with micro-axial flow-pumps resulted in myocardial recovery whereas delayed Impella (mechanical circulatory support) implantation was associated with poor left ventricular recovery. Mechanical circulatory support in patients with refractory cardiogenic shock complicating peripartum cardiomyopathy was associated with a 30-day survival of 100% and a favourable outcome. Notably, early left ventricular unloading combined with bromocriptine therapy was associated with left ventricular recovery. Therefore, an immediate transfer to a tertiary hospital experienced in mechanical circulatory support in combination with bromocriptine treatment seems indispensable for successful treatment of peripartum cardiomyopathy complicated by cardiogenic shock.

16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis.

Many functions of vasoinhibins have been reported, but its receptor has not been clarified yet. Vasoinhibins, 11–18 kDa N-terminal fragments of prolactin, have anti-angiogenic activity and act on endothelial cells to induce apoptosis and to inhibit migration and proliferation, which are opposite to the effects of prolactin. Although vasoinhibins bind to the prolactin receptor, its binding activity is very weak compared to prolactin. Therefore, in this study, we evaluated the binding activity between 16 kDa vasoinhibin and integrin beta1, alpha5 beta1, alpha1 beta1 and alphaV beta3 to identify a specific receptor for vasoinhibins. Moreover, we examined whether 16 kDa vasoinhibin induced apoptosis through integrin beta1 and alpha5 beta1 in endothelial cells. In this study, binding assays and co-immunoprecipitation experiments demonstrated that 16 kDa vasoinhibin could bind strongly to integrin beta1 and alpha5 beta1. Moreover, neutralizing with integrin beta1 and alpha5 beta1 antibody could inhibit 16 kDa vasoinhibin-induced apoptosis in endothelial cells. These findings suggest that vasoinhibins can act on endothelial cells through integrin alpha5 beta1 to induce apoptosis.

Metabolic Syndrome in Hyperprolactinemia.

The metabolic syndrome (MetS) is a conglomerate of clinical findings that convey into increased morbidity and mortality from type 2 diabetes mellitus (T2D) and cardiovascular disease. Hyperprolactinemia (hyperPRL) is associated with components of MetS, especially during pregnancy. Endogenous levels of sex steroids are high during pregnancy in contrast to untreated or replaced hypogonadism in most patients with a prolactinoma and hypogonadism may confer increased risk of MetS in hyperPRL. Dopamine-D2-agonist therapy can improve MetS in patients with a prolactinoma and lower glucose levels in patients with T2D. HyperPRL is a biomarker for decreased dopaminergic tonus in the hypothalamic-pituitary circuit. Patients with a prolactinoma, patients with schizophrenia and/or T2D often have disturbances in this balance and the finding of lower prolactin (PRL) levels in polycystic ovary syndrome (PCOS) may indicate increased dopaminergic tonus. Recent studies supported that PRL levels within or above reference range may be differently related to MetS. In healthy study populations and in PCOS, PRL levels were inversely associated with metabolic risk markers. Ongoing research on PRL fragments, vasoinhibins, may help explain some of the contradicting findings between prolactin levels and metabolism. Improved knowledge about MetS in hyperPRL can characterize subgroups of patients with hyperPRL, who would not otherwise be considered as candidates for dopamine-D2-agonist therapy such as patients with postpartum cardiomyopathy and postmenopausal women with T2D.

Bromocriptine for the Treatment of Peripartum Cardiomyopathy.

Peripartum cardiomyopathy (PPCM) is a life-threatening, pregnancy-associated heart disease that develops towards the end of pregnancy or in the first months following delivery in previously healthy women. Understanding of the pathophysiology has progressed in recent years, highlighting an oxidative-stress mediated cleavage of the nursing hormone prolactin into a toxic 16-kDa prolactin fragment as a major factor driving the disease. The 16-kDa prolactin fragment induces detrimental but potentially reversible effects on heart function. Bromocriptine, a clinically-approved drug to block prolactin release, was initially tested in a PPCM mouse model where it efficiently prevented the onset of PPCM. Consequently, this treatment concept was transferred to and successfully used in humans as a bench-tobedside approach. Encouraging proof-of-concept studies led to a randomised trial that further strengthens the role of bromocriptine in addition to standard heart failure therapy in clinical practice. The aim of this article is to summarise this novel and disease-specific medical treatment, along with current knowledge on the epidemiology and pathophysiology of PPCM.