Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway.

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5ß1 and NF-κB signaling.

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5ß1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1ß, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5ß1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1ß, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5ß1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5ß1 as a promising target for treating vascular inflammation in COVID-19.

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.

Main olfactory bulb reconfiguration by prolonged passive olfactory experience correlates with increased brain-derived neurotrophic factor and improved innate olfaction.

The main olfactory bulb (MOB) is highly plastic and constantly reconfiguring its function and structure depending on sensory experience. Despite the extensive evidence of anatomical, functional and behavioural changes in the olfactory system induced by highly variable olfactory experiences, it is still unknown whether prolonged passive odour experience could reconfigure the MOB at its input and network activity levels and whether these changes impact innate olfaction. Here, by measuring odour-induced glomerular activation, MOB network activity and innate olfactory behaviours, we described a profound MOB reconfiguration induced by prolonged passive olfactory experience in adult animals that impacts MOB input integration at the glomerular layer including an increase in the activated glomerular area and signal intensity, which is combined with a refinement in the number of activated glomeruli and less-overlapped glomerular maps. We also found that prolonged passive olfactory experience dramatically changes MOB population activity in the presence and absence of odours, which is reflected as a decrease in slow oscillations (<12 Hz) and an increase in fast oscillations (>12 Hz). All these functional changes in awake and anaesthetized mice correlate with an increase in brain-derived neurotrophic factor (BDNF) and with improved innate olfactory responses such as habituation/dishabituation and innate preference/avoidance. Our study shows that prolonged passive olfactory experience in adult animals produces a dramatic reconfiguration of the MOB network, possibly driven by BDNF, that improves innate olfactory responses.

A comparative phylogenetic analysis of prolactin cleavage sites for the generation of vasoinhibin in vertebrates.

Vasoinhibin is a pleiotropic protein hormone with endocrine, autocrine, and paracrine effects on blood vessel growth, permeability, and dilation, and a role in several human diseases. It is generated by proteolytic cleavage of the pituitary hormone prolactin by cathepsin D. Several isoforms with a variation in the number of amino acids and corresponding molecular mass exist. This in silico study investigated the cathepsin D cleavage sites in prolactin responsible for the generation of vasoinhibin in vertebrate species. Ninety-one prolactin protein sequences from species of the taxa primates, rodents, laurasiatheria, mammals, sauropsida, and fish were retrieved, and a multiple sequence alignment was performed. Each sequence was investigated for the presence of a vasoinhibin-generating cathepsin D cleavage site and its corresponding substrate affinity using a scoring system. Primates demonstrated the highest substrate affinity for the generation of the 15 kDa vasoinhibin isoform, and fish the highest affinity for the 16.8 kDa isoform. In both cases, this associates to the presence of leucine in the cleavage site, which is not present in species of the other taxa. In primate evolution, the presence of leucine in the cleavage site occurs with the emergence of simiiformes 42 million years ago and is conserved in higher primates across all subsequent speciation nodes. The 17.2 kDa vasoinhibin isoform has a constant substrate affinity in all taxa. The presence of leucine in vasoinhibin generating cleavage sites appears as an important feature of the molecular evolution of vasoinhibin.

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

Increased 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-1ß, 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.

Vasoinhibin generation and effect on neuronal apoptosis in the hippocampus of late mouse embryos.

Morphological and behavioral evidence suggests that vasoinhibin is present in the central nervous system (CNS), triggering neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin reduces neuronal survival and differentiation of primary sensory neurons of the peripheral nervous system. To address the functional role played by vasoinhibin at the CNS, and to better understand the underlying mechanisms involved in its actions, we treated primary cultured hippocampal neurons obtained from embryonic day 16 (E16) mice with a human recombinant vasoinhibin. We examined the resulting cellular changes, focusing on neuronal cell death, and explored the local generation of vasoinhibin within the hippocampus. Our results show that vasoinhibin significantly reduced neuronal cell density and increased immunoreactive activated caspase-3 and TUNEL-positive staining at 72, 16, and 24 h, respectively. Furthermore, vasoinhibin increased the expression of proapoptotic genes BAX, BAD, BIM, and PUMA and decreased that of the antiapoptotic gene BCL-2 at 24 h, as assessed by quantitative real-time reverse transcription-polymerase chain reaction. Vasoinhibin effects were blocked by coincubation with a vasoinhibin antibody or with prolactin. Immunoreactive bands consistent with vasoinhibin were observed in hippocampal extracts by Western blot analysis, and a prolactin standard was cleaved to vasoinhibin by a hippocampal lysate in a heat- and cathepsin D inhibitor pepstatin A-dependent fashion. Taken together, these data support the notion that vasoinhibin is locally produced by cathepsin D within the embryonic mouse hippocampus, a brain region that plays a critical role in emotional regulation, resulting in decreased neuronal cell viability via the activation of the intrinsic apoptosis pathway.

Matrix Metalloproteases and Cathepsin D in Human Serum do not Cleave Prolactin to Generate Vasoinhibin.

BACKGROUND: Vasoinhibin is generated in the pituitary gland and in multiple target tissues by proteolytic cleavage of prolactin by matrix metalloproteinases and cathepsin D. A dysregulation of vasoinhibin generation appears to contribute to diabetic retinopathy and diabetic macular edema, retinopathy of prematurity, peripartum cardiomyopathy, and preeclampsia. Here, we investigate whether vasoinhibin is generated by matrix metalloproteinases and cathepsin D in human serum. METHODS: The abundance of matrix metalloproteinases 1, 2, 3, 8, 9, 10, 13, tissue inhibitors of metalloproteinases 1, 2, 4, and the activity of cathepsin D in serum samples were determined. Samples from healthy male (n = 3) and female (n = 2) subjects, pregnant subjects (n = 2), and patients with type 2 diabetes mellitus (n = 2) were investigated. The samples were incubated with recombinant prolactin at 37°C, under different pH, time, and buffer conditions. Prolactin and cleaved prolactin products were investigated by SDS-PAGE and western blotting. RESULTS: Matrix metalloproteases-1, -2, -3, -8, -9, -10, -13, TIMP-1, -2, and -4, and the activity of cathepsin D were detected in all sera. Full-length prolactin incubated with human sera, containing endogenous matrix metalloproteinases and cathepsin D, remained intact at neutral pH during a time frame from 1 to 24 hours. Partial enzymatic cleavage of prolactin resulting in the generation of a vasoinhibin-like 17 kDa peptide was observed in samples incubated at pH 3.4. Heat inactivation of the serum and the addition of an MMP inhibitor suppressed the generation of the 17 kDa peptide, indicating that its generation was MMP-mediated. CONCLUSIONS: Vasoinhibin generation by enzymatic cleavage of prolactin by matrix metalloproteases or cathepsin D does not occur in human serum at physiological pH. A limited proteolysis of prolactin, resulting in the generation of a vasoinhibin-like peptide with an apparent molecular weight of 17 kDa occurs in serum at acidic pH. The generation of vasoinhibin may require the cellular and tissue microenvironments.

Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Maternal diet during lactation affects offspring metabolic health throughout life. Prolactin (PRL) is present in high quantities in maternal milk; however, the effects of milk PRL on the offspring remain poorly characterized. In this study, we evaluated whether feeding a high-fat diet (HFD) to rats during lactation alters PRL, both in the mother's serum and in milk, and whether this factor contributes to HFD-induced metabolic dysfunction in the offspring. Maternal HFD resulted in decreased PRL levels in milk (but not in serum), reduced mammary gland (MG) PRL receptor expression, and altered MG structure and function. Offspring from HFD-fed dams had increased body weight and adiposity, and developed fatty liver, hyperinsulinemia, and insulin resistance at weaning. Increasing PRL levels in the HFD-fed mothers by subcutaneous osmotic minipumps releasing PRL normalized MG function and PRL levels in milk. Moreover, PRL treatment in HFD-fed mothers, or directly in their pups via oral PRL administration, increased liver STAT5 phosphorylation, reduced visceral adiposity, ameliorated fatty liver, and improved insulin sensitivity in offspring. Our results show that HFD impairs PRL actions during lactation to negatively affect MG physiology and directly impair offspring metabolism.-De los Ríos, E. A., Ruiz-Herrera, X., Tinoco-Pantoja, V., López-Barrera, F., Martínez de la Escalera, G., Clapp, C., Macotela, Y. Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Sequence optimization and glycosylation of vasoinhibin: Pitfalls of recombinant production.

Vasoinhibin belongs to a family of proteins with antiangiogenic properties derived by proteolytic cleavage from the hormone prolactin (PRL). Vasoinhibin isoforms range from the first 79 to the first 159 residues of PRL. In an attempt to increase the yield of recombinant vasoinhibin and avoid incorrect intra- and inter-disulfide bond formation, the cDNA sequence comprising the first 123 amino acids of human PRL, in which cysteine 58 was or not mutated to serine, was codon-optimized. The optimized constructs achieved a 6-fold increase in mRNA expression but showed no change in protein production and reduced protein secretion when expressed in human embryo kidney (HEK293T/17) cells. Limited vasoinhibin levels associated with the activation of the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD) as revealed by the upregulation of UPR (Bip, Xbp-1, and Chop) and ERAD (Hrd1, Os9, and Sel1l) target genes. Mutation to serine introduced a new N-glycosylation site and associated with increased glycosylation and release of glycosylated vasoinhibin isoforms having reduced antiangiogenic properties. We conclude that overexpression and excessive glycosylation lead to protein degradation and reduced bioactivity, respectively, negatively affecting the production of recombinant vasoinhibin in mammalian cells.

Vasoinhibin Suppresses Nerve Growth Factor-Induced Differentiation and Survival of PC12 Pheochromocytoma Cells.

BACKGROUND: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells. METHODS: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting. RESULTS: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells. CONCLUSIONS: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation.

Lack of Delta-Sarcoglycan (Sgcd) Results in Retinal Degeneration.

Age-related macular degeneration (AMD) is the leading cause of central vision loss and severe blindness among the elderly population. Recently, we reported on the association of the SGCD gene (encoding for δ-sarcoglycan) polymorphisms with AMD. However, the functional consequence of Sgcd alterations in retinal degeneration is not known. Herein, we characterized changes in the retina of the Sgcd knocked-out mouse (KO, Sgcd-/-). At baseline, we analyzed the retina structure of three-month-old wild-type (WT, Sgcd+/+) and Sgcd-/- mice by hematoxylin and eosin (H&E) staining, assessed the Sgcd-protein complex (α-, ß-, γ-, and ε-sarcoglycan, and sarcospan) by immunofluorescence (IF) and Western blot (WB), and performed electroretinography. Compared to the WT, Sgcd-/- mice are five times more likely to have retinal ruptures. Additionally, all the retinal layers are significantly thinner, more so in the inner plexiform layer (IPL). In addition, the number of nuclei in the KO versus the WT is ever so slightly increased. WT mice express Sgcd-protein partners in specific retinal layers, and as expected, KO mice have decreased or no protein expression, with a significant increase in the α subunit. At three months of age, there were no significant differences in the scotopic electroretinographic responses, regarding both a- and b-waves. According to our data, Sgcd-/- has a phenotype that is compatible with retinal degeneration.

Prolactin regulates liver growth during postnatal development in mice.

The liver grows during the early postnatal period first at slower and then at faster rates than the body to achieve the adult liver-to-body weight ratio (LBW), a constant reflecting liver health. The hormone prolactin (PRL) stimulates adult liver growth and regeneration, and its levels are high in the circulation of newborn infants, but whether PRL plays a role in neonatal liver growth is unknown. Here, we show that the liver produces PRL and upregulates the PRL receptor in mice during the first 2 wk after birth, when liver growth lags behind body growth. At postnatal week 4, the production of PRL by the liver ceases coinciding with the elevation of circulating PRL and the faster liver growth that catches up with body growth. PRL receptor null mice ( Prlr-/-) show a significant decrease in the LBW at 1, 4, 6, and 10 postnatal weeks and reduced liver expression of proliferation [cyclin D1 ( Ccnd1)] and angiogenesis [platelet/endothelial cell adhesion molecule 1 ( Pecam1)] markers relative to Prlr+/+ mice. However, the LBW increases in Prlr-/- mice at postnatal week 2 concurring with the enhanced liver expression of Igf-1 and the liver upregulation and downregulation of suppressor of cytokine signaling 2 ( Socs2) and Socs3, respectively. These findings indicate that PRL acts locally and systemically to restrict and stimulate postnatal liver growth. PRL inhibits liver and body growth by attenuating growth hormone-induced Igf-1 liver expression via Socs2 and Socs3-related mechanisms.

Vasoinhibin Suppresses the Neurotrophic Effects of VEGF and NGF in Newborn Rat Primary Sensory Neurons.

BACKGROUND/AIMS: Studies on the biological actions of vasoinhibins have focused mainly on endothelial cells. However, there is incipient knowledge about how vasoinhibins affect the nervous system, even if the target cells and mechanisms of action involved in these effects are unknown. METHODS: In order to determine if neurons are direct targets of vasoinhibins, we examined cellular outcomes and the intracellular pathways involved in the neuronal actions of vasoinhibins using newborn rat dorsal root ganglion (DRG) neurons as a model system. RESULTS: Vascular endothelial growth factor (VEGF) or nerve growth factor (NGF) treatment for 48 h resulted in neurite outgrowth stimulation in both DRG cultured explants and isolated primary sensory neurons. Interestingly, a recombinant vasoinhibin containing the first 123 amino acids of human prolactin antagonized the VEGF- and NGF-induced stimulation of neurite outgrowth. Vasoinhibin significantly reduced the density of neurites in DRG explants and obliterated neuritogenesis in isolated DRG neurons in primary culture, supporting a direct neuronal effect of vasoinhibin. In cultures of isolated DRG cells, virtually all ß3-tubulin-labeled cells express TrkA, and the majority of these cells also express VEGFR2. Short-term VEGF or NGF treatment of DRG explants resulted in increased ERK1/2 and AKT phosphorylation, whereas incubation of DRG with the combination of either VEGF or NGF together with vasoinhibin resulted in blunted VEGF- or NGF-induced phosphorylation of both ERK1/2 and AKT. CONCLUSION: Our results show that primary sensory neurons are direct targets of vasoinhibin, and suggest that vasoinhibin inhibition of neurite outgrowth involves the disruption of ERK and AKT phosphorylation cascades.

Higher prolactin and vasoinhibin serum levels associated with incidence and progression of retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity (ROP) is a potentially blinding, retinal neovascular disease. Systemic prolactin accesses the retina to regulate blood vessels. Prolactin is proangiogenic and can be cleaved to antiangiogenic vasoinhibins. We investigated whether circulating prolactin and vasoinhibins associate with incidence and progression of ROP. METHODS: A prospective, longitudinal, case-control study covering postnatal weeks 1 to 9 measured serum prolactin, vasoinhibins, and vascular endothelial growth factor (VEGF) weekly in 90 premature infants diagnosed as ROP or control. RESULTS: Prolactin levels were higher in ROP than in control patients before (106.2 ± 11.3 (SEM) vs. 64.7 ± 4.9 ng/ml, postnatal week 1) and during (120.6 ± 10 vs. 84.7 ± 7.5ng/ml, postnatal week 5) ROP diagnosis. Prolactin, but not gestational age, birth weight, Apgar score, sepsis, or ventilation time, correlated with ROP. The relative risk (RR) of developing ROP increased if Prolactin (PRL) levels were higher than thresholds of 80 ng/ml (RR = 1.55, 95% CI: 1.06-2.28), 100 ng/ml (RR = 1.63, 95% CI: 1.14-2.34), or 120 ng/ml (RR = 1.95, 95% CI: 1.41-2.68). Vasoinhibin levels were 39.7% higher (95% CI: 4.5-77.5) in the circulation of ROP than in control patients at postnatal week 1 and similar thereafter, whereas VEGF serum levels were always similar. CONCLUSION: High serum prolactin and vasoinhibin levels predict and may impact ROP progression.

The role of the prolactin/vasoinhibin axis in rheumatoid arthritis: an integrative overview.

Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease destroying articular cartilage and bone. The female preponderance and the influence of reproductive states in RA have long linked this disease to sexually dimorphic, reproductive hormones such as prolactin (PRL). PRL has immune-enhancing properties and increases in the circulation of some patients with RA. However, PRL also suppresses the immune system, stimulates the formation and survival of joint tissues, acquires antiangiogenic properties upon its cleavage to vasoinhibins, and protects against joint destruction and inflammation in the adjuvant-induced model of RA. This review addresses risk factors for RA linked to PRL, the effects of PRL and vasoinhibins on joint tissues, blood vessels, and immune cells, and the clinical and experimental data associating PRL with RA. This information provides important insights into the pathophysiology of RA and highlights protective actions of the PRL/vasoinhibin axis that could lead to therapeutic benefits.

Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1.

Adeno-associated virus (AAV) vector-mediated delivery of inhibitors of blood-retinal barrier breakdown (BRBB) offers promise for the treatment of diabetic macular edema. Here, we demonstrated a reversal of blood-retinal barrier pathology mediated by AAV type 2 (AAV2) vectors encoding vasoinhibin or soluble VEGF receptor 1 (sFlt-1) when administered intravitreally to diabetic rats. Efficacy and safety of the AAV2 vasoinhibin vector were tested by monitoring its effect on diabetes-induced changes in the retinal vascular bed and thickness, and in the electroretinogram (ERG). Also, the transduction of AAV2 vectors and expression of AAV2 receptors and co-receptors were compared between the diabetic and the non-diabetic rat retinas. AAV2 vasoinhibin or AAV2 sFlt-1 vectors were injected intravitreally before or after enhanced BRBB due to diabetes induced by streptozotocin. The BRBB was examined by the Evans blue method, the vascular bed by fluorescein angiography, expression of the AAV2 EGFP reporter vector by confocal microscopy, and the AAV2 genome, expression of transgenes, receptors, and co-receptors by quantitative PCR. AAV2 vasoinhibin and sFlt-1 vectors inhibited the diabetes-mediated increase in BRBB when injected after, but not before, diabetes was induced. The AAV2 vasoinhibin vector decreased retinal microvascular abnormalities and the diabetes-induced reduction of the B-wave of the ERG, but it had no effect in non-diabetic controls. Also, retinal thickness was not altered by diabetes or by the AAV2 vasoinhibin vector. The AAV2 genome, vasoinhibin and sFlt-1 transgenes, and EGFP levels were higher in the retinas from diabetic rats and were associated with an elevated expression of AAV2 receptors (syndecan, glypican, and perlecan) and co-receptors (fibroblast growth factor receptor 1, αvß5 integrin, and hepatocyte growth factor receptor). We conclude that retinal transduction and efficacy of AAV2 vectors are enhanced in diabetes, possibly due to their elevated cell entry. AAV2 vectors encoding vasoinhibin and sFlt-1 may be desirable gene therapeutics to target diabetic retinopathy and macular edema.

The hormone prolactin is a novel, endogenous trophic factor able to regulate reactive glia and to limit retinal degeneration.

Retinal degeneration is characterized by the progressive destruction of retinal cells, causing the deterioration and eventual loss of vision. We explored whether the hormone prolactin provides trophic support to retinal cells, thus protecting the retina from degenerative pressure. Inducing hyperprolactinemia limited photoreceptor apoptosis, gliosis, and changes in neurotrophin expression, and it preserved the photoresponse in the phototoxicity model of retinal degeneration, in which continuous exposure of rats to bright light leads to retinal cell death and retinal dysfunction. In this model, the expression levels of prolactin receptors in the retina were upregulated. Moreover, retinas from prolactin receptor-deficient mice exhibited photoresponsive dysfunction and gliosis that correlated with decreased levels of retinal bFGF, GDNF, and BDNF. Collectively, these data unveiled prolactin as a retinal trophic factor that may regulate glial-neuronal cell interactions and is a potential therapeutic molecule against retinal degeneration.

Principles of the prolactin/vasoinhibin axis.

The hormonal family of vasoinhibins, which derive from the anterior pituitary hormone prolactin, are known for their inhibiting effects on blood vessel growth, vasopermeability, and vasodilation. As pleiotropic hormones, vasoinhibins act in multiple target organs and tissues. The generation, secretion, and regulation of vasoinhibins are embedded into the organizational principle of an axis, which integrates the hypothalamus, the pituitary, and the target tissue microenvironment. This axis is designated as the prolactin/vasoinhibin axis. Disturbances of the prolactin/vasoinhibin axis are associated with the pathogenesis of retinal and cardiac diseases and with diseases occurring during pregnancy. New phylogenetical, physiological, and clinical implications are discussed.

Prolactin anterior pituitary expression and circulating levels are reduced in obese and diabetic rats: role of TGF-ß and TNF-α.

The levels of the hormone prolactin (PRL) are reduced in the circulation of patients with Type 2 diabetes and in obese children, and lower systemic PRL levels correlate with an increased prevalence of diabetes and a higher risk of metabolic syndrome. The secretion of anterior pituitary (AP) PRL in metabolic diseases may be influenced by the interplay between transforming growth factor ß (TGF-ß) and tumor necrosis factor α (TNF-α), which inhibit and can stimulate AP PRL synthesis, respectively, and are known contributors to insulin resistance and metabolic complications. Here, we show that TGF-ß and TNF-α antagonize the effect of each other on the expression and release of PRL by the GH4C1 lactotrope cell line. The levels of AP mRNA and circulating PRL decrease in high-fat diet-induced obese rats in parallel with increased and reduced AP levels of TGF-ß and TNF-α mRNA, respectively. Likewise, AP expression and circulating levels of PRL are reduced in streptozotocin-induced diabetic rats and are associated with higher AP expression and protein levels of TGF-ß and TNF-α. The opposing effects of the two cytokines on cultured AP cells, together with their altered expression in the AP of obese and diabetic rats suggest they are linked to the reduced PRL production and secretion characteristics of metabolic diseases.