Increased angiogenesis and vascular permeability are hallmarks of microvascular retinal diseases such as diabetic retinopathy and diabetic macular edema (DME). Periodic intravitreal injections of inhibitors of the vascular endothelial growth factor (VEGF) are first-line therapy, but their invasiveness and associated risks often lead to poor compliance and outcomes. Here, we investigate VIAN-c4551, a highly potent antiangiogenic cyclic heptapeptide, as a non-invasive topical ophthalmic alternative to the current standard of care for DME. VIAN-c4551 demonstrated high potency (IC50 = 137 pM) to inhibit the permeability of human umbilical vein endothelial cell monolayers induced by VEGF. VIAN-c4551 eye drops potently (0.005% minimum effective dose) prevented, for up to 24 h, the retinal vascular leakage induced by VEGF injected intravitreally and reversed the increased retinal vascular permeability due to diabetes in rats and mice. VIAN-c4551 exhibited high penetrability across MDCK epithelium and, after a single eye drop in rabbits, reached the vitreous and the retina-choroid at concentrations several orders of magnitude above its IC50 (Cmax ~239 nM and ~ 6.7 µM, respectively, after 6 h) that lasted at least 24 h. In conclusion, VIAN-c4551 is a non-invasive, once-a-day potential intervention for preventing and reversing retinal vascular leakage in DME and other vascular retinopathies and preserving sight.

Reactive oxygen species (ROS) are physiological byproducts of neuronal metabolism. However, an imbalance between ROS generation and antioxidant capacity, often driven by dysregulated pro-oxidant enzymes like nicotinamide adenine dinucleotide phosphate oxidases (NOX), can result in deleterious oxidative stress. This oxidative stress is a critical factor in the pathogenesis of neurodegenerative diseases. While interventions with broad-spectrum antioxidants have demonstrated limited efficacy, the modulation of endogenous antioxidant mechanisms presents a promising therapeutic avenue. Here, we investigated the potential of the neuroprotective hormone prolactin to mitigate oxidative stress and subsequent neuronal cell death. Prolactin protected primary mouse hippocampal neurons from hydrogen peroxide (H2O2)-induced oxidative damage. Prolactin reduced ROS levels, lipid peroxidation, and apoptosis, and its effects were occluded by a specific prolactin receptor antagonist (G129R-hPRL). Mechanistically, prolactin suppressed H2O2-induced mRNA upregulation of pro-oxidative Nox4 and pro-apoptotic Bax. Moreover, prolactin induced nuclear factor kappa B (NF-κB) nuclear translocation, and the inhibition of the NF-κB signaling pathway abolished the neuroprotective and transcriptional effects of prolactin, indicating its central role in prolactin-mediated protection. Our findings indicate that prolactin exerts potent antioxidant and neuroprotective effects by modulating the expression of Nox4 and Bax, thereby reducing ROS generation and neuronal apoptosis. This study underscores the therapeutic potential of prolactin in attenuating oxidative stress and suggests a possible role in the treatment of neurodegenerative diseases.

Ischemic stroke is a significant global health issue, ranking fifth among all causes of death and a leading cause of serious long‐term disability. Ischemic stroke leads to severe outcomes, including permanent brain damage and neuronal dysfunction. Therefore, decreasing and preventing neuronal injuries caused by stroke has been the focus of therapeutic research. In recent years, many studies have shown that fluctuations in hormonal levels influence the prognosis of ischemic stroke. Thus, it is relevant to understand the role of hormones in the pathophysiological mechanisms of ischemic stroke for preventing and treating this health issue. Here, we investigate the contribution of the prolactin/vasoinhibin axis, an endocrine system regulating blood vessel growth, immune processes, and neuronal survival, to the pathophysiology of ischemic stroke. Male mice with brain overexpression of prolactin or vasoinhibin by adeno‐associated virus (AAV) intracerebroventricular injection or lacking the prolactin receptor ( Prlr −/− ) were exposed to transient middle cerebral artery occlusion (tMCAO) for 45 min followed by 48 h of reperfusion. Overexpression of vasoinhibin or the absence of the prolactin receptor led to an increased lesion volume and decreased survival rates in mice following tMCAO, whereas overexpression of prolactin had no effect. In addition, astrocytic distribution in the penumbra was altered, glial fibrillary acidic protein and S100b mRNA expressions were reduced, and interleukin‐6 mRNA expression increased in the ischemic hemisphere of mice overexpressing vasoinhibin. Of note, prolactin receptor‐null mice ( Prlr −/− ) showed a marked increase in serum vasoinhibin levels. Furthermore, vasoinhibin decreased astrocyte numbers in mixed hippocampal neuron–glia cultures. These observations suggest that increased vasoinhibin levels may hinder astrocytes' protective reactivity. Overall, this study suggests the involvement of the prolactin/vasoinhibin axis in the pathophysiology of ischemic stroke‐induced brain injury and provides insights into the impact of its dysregulation on astrocyte reactivity and lesion size. Understanding these mechanisms could help develop therapeutic interventions in ischemic stroke and other related neurological disorders.

Basal electroretinogram (ERG) oscillations have shown predictive value for modifiable risk factors for type 2 diabetes. However, their origin remains unknown. Here, we seek to establish the pharmacological profile of the low delta-like (δ1) wave in the mouse because it shows light sensitivity in the form of a decreased peak frequency upon photopic exposure. Applying neuropharmacological drugs by intravitreal injection, we eliminated the δ1 wave using lidocaine or by blocking all chemical and electrical synapses. The δ1 wave was insensitive to the blockade of photoreceptor input, but was accelerated when all inhibitory or ionotropic inhibitory receptors in the retina were antagonized. The sole blockade of GABAA, GABAB, GABAC, and glycine receptors also accelerated the δ1 wave. In contrast, the gap junction blockade slowed the δ1 wave. Both GABAA receptors and gap junctions contribute to the light sensitivity of the δ1 wave. We further found that the day light-activated neuromodulators dopamine and nitric oxide donors mimicked the effect of photopic exposure on the δ1 wave. All drug effects were validated through light flash-evoked ERG responses. Our data indicate that the low δ-like intrinsic wave detected by the non-photic ERG arises from an inner retinal circuit regulated by inhibitory neurotransmission and nitric oxide/dopamine-sensitive gap junction-mediated communication.

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.

En las reacciones bioquímicas del metabolismo donde participa el oxígeno molecular se presenta una constante formación de especies reactivas de oxígeno (ROS). Entre las diferentes ROS, destaca el peróxido de hidrógeno (H2O2) por su papel en la fisiología celular, ya que participa como segundo mensajero en múltiples procesos celulares como la proliferación celular, la síntesis y la secreción de hormonas, la regulación y la activación de células inmunes, la angiogénesis y los procesos apoptóticos. Sin embargo, un aumento exacerbado en la formación y la acumulación de H2O2, producido por el incremento en la actividad de la cadena transportadora de electrones en la mitocondria, la autooxidación de la glucosa y/o la activación de la vía del sorbitol, genera un desequilibrio en el estado de óxido-reducción de las células. Esta acumulación de H2O2 puede causar un daño celular a través de la oxidación de las proteínas, los lípidos, los carbohidratos y los ácidos nucleicos; al contribuir en el desarrollo y la progresión de enfermedades degenerativas y metabólicas como la diabetes mellitus. Al subir la concentración de la glucosa en la sangre, se presenta un incremento en la concentración del H2O2, que intensifica los procesos oxidantes que están ligados a la patogénesis, la progresión y las complicaciones de la diabetes mellitus.

The progression of amyloid plaques and neurofibrillary tangles in different brain areas is associated with the effects of Alzheimer’s disease (AD). In addition to cognitive impairment, circadian alterations in locomotor activity have also been detected, but they have not been characterized in a jet lag protocol. Therefore, the present study aimed to compare 3xTg-AD and non-transgenic mice in changes of 24 h cycles of spontaneous locomotor activity in a jet lag protocol, in an environment without a running wheel, at 3 different states of neuronal damage: early, intermediate and advanced (3, 8 and 13 months, respectively). The 3xTg-AD mice at 3 months presented differences in phase angle and acrophase, and differentially increased activity after advances more than after delays. At 13 months, a shortening of the free-running period in constant darkness was also noted. 3xTg-AD mice showed a significant increase (123%) in global activity at 8 to 13 months and in nighttime activity (153%) at 13 months. In the advance protocol (ADV), 3xTg-AD mice displayed a significant increase in global activity (171%) at 8 and 13 months. The differences in masking effect were evident at 8 months. To assess a possible retinal dysfunction that could interfere with photic entrainment as part of the neurodegenerative process, we compared electroretinogram recordings. The results showed early deterioration in the retinal response to light flashes in mesopic conditions, observed in the B-wave latency and amplitude. Thus, our study presents new behavioral and pathological characteristics of 3xTg-AD mice and reveals the usefulness of noninvasive tools in early diagnosis.

We report here that the pituitary hormone prolactin (PRL) has an important, conserved role in regulating adrenal gland function. The adrenal plays a pivotal role in endocrine homeostasis and stress response, which vary across lifespan and have different features in the two sexes.[1] Furthermore, most adrenal disorders have a higher prevalence in women.[2] To get deeper insight into the mechanisms of age- and sex-dependent adrenal function, we performed an integrative analysis of the mouse adrenal transcriptome by RNA-seq and active enhancer (as defined by H3K27ac ChIP-seq) usage at different ages (from E18.5 to P12 weeks) in both sexes. Multivariate analysis showed that age, but not sex, had a significant effect on adrenal global gene expression profiles (Figure and Supporting information S1; Tables S1, S2). Expression of genes related to lipid metabolism and immunity was progressively enriched with age (Figure 1C and Supporting information Table S3), with the proportion of macrophages increasing and neutrophils/mast cells decreasing at older ages (Figure 1D). The expression of different classes of secreted proteins, ion channels and enzymes showed a lower level of sex-dependent enrichment (Figure 1E and Supporting information Table S4). Age-and sex-dependent long non-coding RNA differentially expressed genes (DEG) followed the same pattern as coding DEG, being mostly specific for each age and sex (Supporting information Figure S2 and Table S5). Remarkably, P12 weeks adrenals of both sexes showed increased percentages of novel gene transcripts compared to previous ages (Supporting information Figure S2E). Sexually dimorphic expression pervades all cell populations of the mouse adrenal gland (Supporting information Figure S3 and Table S6). We identified a few thousand enhancers active in the adrenal gland at each temporal stage and in each sex (Supporting information Table S7). A subset among those belong to the super-enhancer class (SEC), genomic regions highly enriched in active chromatin, which regulate developmental and tissue-specific programs and often encompass disease-associated genomic loci.[3] At all ages and in both sexes, adrenal SEC have a much higher level of overlap than typical-enhancer class (TEC) elements (Figure 2A), while adrenal TEC are significantly more conserved than SEC (Figure 2B). Adrenal SEC-associated genes are enriched in genes involved in transcriptional regulation, cell-cell adhesion, protein phosphorylation, biological rhythms and steroid hormone receptor function at all ages and in both sexes (Figure 2C and Supporting information Table S8). Mouse adrenal SEC-associated genes are expressed at significantly higher levels in the adrenal gland (Supporting information Figure S4A) and include a higher percentage of tissue-specific genes (Supporting information Figure S4B) than TEC-associated genes. Human adrenal SEC are also preferentially associated to genes encoding proteins with adrenal-enriched expression than to genes with higher expression in other tissues (Supporting information Figure S4C) and SEC-associated genes are enriched with age-dependent DEG at most times and sex-dependent DEG at P12 weeks compared to TEC (Supporting information Figure S4D,E). Examples of SEC associated to genes important in adrenal physiology are shown in Supporting information Figure S5. Of note, some adrenal SEC-associated genes are expressed preferentially in the adrenal and are involved in blood pressure regulation (Supporting information Table S9). In particular, non-coding SNPs associated with blood pressure traits with very high significance are located inside a conserved SEC encompassing the KCNK3 gene (Supporting information Figure S6 and Table S10). By comparing our list of adrenal sexually dimorphic DEG with data in mouse[4, 5] and rat,[6] we could highlight a core conserved sexually dimorphic gene expression program (Figure 3A and Supporting information Table S11). We focused on the role of the PRL receptor (PRLR), because of its conserved sexually dimorphic expression in the human adrenal (Figure 3B) and the well-known role of PRL signalling in physiological adaptations and response to stress.[7] All Prlr isoforms were upregulated in the female adrenal compared to male at P12, but not P2 weeks of age (Figure 3C). Adrenal gland weight was significantly reduced in adult female Prlr -/- mice compared to WT (Figure 3D), while total body weight was normal in all Prlr -/- animals (Supporting information Figure S7). Signalling pathway impact analysis revealed that the JAK-STAT pathway is significantly activated in female adrenals compared to male at P12 weeks of age (Supporting information Figure S8). The mean area of adrenocortical cells in female Prlr -/- adrenals was significantly decreased (Figure 3E,F). This is consistent with significantly reduced circulating corticosterone levels and a trend toward an increased ACTH/corticosterone ratio in female Prlr -/- mice compared to WT (Figure 3G,H). These results show that PRL signalling through the PRLR has a crucial role in shaping the sexually dimorphic mouse adrenal phenotype. To assess the translational relevance of the mouse model results and to investigate the role of prolactin signalling in human physiopathology, we compared circulating adrenal steroid hormone levels in patients with PRL-secreting (prolactinoma; PRLA) and non-functioning pituitary adenomas (NFPA) (Supporting information Figure S9 and Table S12). Dehydroepiandrosterone sulphate (DHEAS) levels were significantly higher in patients with PRLA compared to NFPA (Figure 4A). In the PRLA group, men had higher circulating PRL levels than women (Figure 4B) and the PRL/DHEAS ratio was significantly more elevated in men than in women (Figure 4C). DHEAS levels were significantly reduced after therapy with dopamine agonists which inhibit PRL, but not ACTH, secretion (Figure 4D-F). Overall, these data suggest that the female adrenal may be more sensitive to PRL effects also in humans. This parallels what we have shown in mice in this study and points on the sexual dimorphic expression of PRLR in the human adrenal gland as a key component of this increased response.[1, 8] In humans, the effect of high PRL to preferentially increase DHEAS levels may be related to the enriched expression of PRLR in the adrenocortical zona reticularis.[9] Modulation of adrenal steroid secretion by PRL may, thus, contribute to the positive effects of physiological concentrations of this hormone on metabolic homeostasis in basal conditions and under stress, while its deregulation in hypo- and hyperprolactinemic states may play an important role in the clinical manifestations of PRL deficiency or excess, respectively. In conclusion, we have unveiled a crucial role for PRL signalling in the sexually dimorphic phenotype of the adult adrenal gland. Our results open new perspectives for the therapy of disorders characterized by adrenal hormones hypersecretion through the use of drugs modulating prolactin release. We thank Frédéric Brau for plugin development for image analysis, Marcin Rucinski for sharing rat adrenal gland microarray data, Xarubet Ruiz-Herrera for animal breeding and genotyping, Matthias Kroiss and Bonald Figueiredo for discussions and critical reading of the manuscript. Funding: This study was supported by the Fondation ARC Project PJA 20191209289 grant to C.R., grants from the Agence Nationale de la Recherche (ANR) ANR15-CE14-0017 (LOCALDO) and ANR20-CE14-0007 (Goldilocks), CNRS EXPOGEN-CANCER International Research Project, Fondation Jérôme Lejeune #1944 to E.L and by the Deutsche Forschungsgemeinschaft (DFG) within the CRC/Transregio (project number: 314061271). The Galaxy server that was used for data analysis is in part funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry of Education and Research [BMBF grants 031 A538A/A538C RBC, 031L0101B/031L0101C de.NBI-epi, 031L0106 de.STAIR (de.NBI)]. The authors declare that they have no conflict of interest. Conceptualization: E.L.; methodology: C.R, E.A., L. S.-M., M.D.-B., N.D., M.J., M.K.; data analysis: C.R., B.A., M.D., F.C., T.D, C.C, E.L.; supervision: T.D., C.C., E.L; writing-original draft: E.L; writing-review and editing: all authors. RNA-seq data: Gene Expression Omnibus GSE173691 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE173691). ChIP-seq data: Gene Expression Omnibus GSE173704 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE173704). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

Aging causes the progressive degeneration of retinal cells leading to the eventual loss of vision. The hormone prolactin (PRL) is a neurotrophic factor able to compensate for photoreceptor cell death and electroretinogram deficits induced by light retinal damage. Here, we used adult 4-month old and aged 20-month old pigmented mice, null or not for the PRL receptor to explore whether PRL provides trophic support against age-related retinal dysfunction. Retinal functionality, apoptosis, glia activation, and neurotrophin expression were assessed by electroretinogram, TUNEL, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 immunohistochemistry, and real-time PCR, respectively. Lack of PRL signaling in aged mice, but not in adult mice, correlated with photosensitive retinal dysfunction, increased photoreceptor apoptosis, differential expression of proapoptotic mediators, and microglia activation. We conclude that PRL is required for maintaining retinal functionality in both female and male mice during aging and has potential therapeutic value against age-related retinal disorders.

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.

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 β3-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.

Excessive accumulation of body fat triggers insulin resistance and features of the metabolic syndrome. Recently, evidence has accumulated that obesity, type 2 diabetes, and metabolic syndrome are associated with reduced levels of serum prolactin (PRL) in humans and rodents, raising the question of whether low PRL levels contribute to metabolic dysfunction. Here, we have addressed this question by investigating the role of PRL in insulin sensitivity and adipose tissue fitness in obese rodents and humans. In diet-induced obese rats, treatment with PRL delivered via osmotic mini-pumps, improved insulin sensitivity, prevented adipocyte hypertrophy, and reduced inflammatory cytokine expression in visceral fat. PRL also induced increased expression of Pparg and Xbp1s in visceral adipose tissue and elevated circulating adiponectin levels. Conversely, PRL receptor null mice challenged with a high-fat diet developed greater insulin resistance, glucose intolerance, and increased adipocyte hypertrophy compared with wild-type mice. In humans, serum PRL values correlated positively with systemic adiponectin levels and were reduced in insulin-resistant patients. Furthermore, PRL circulating levels and PRL produced by adipose tissue correlated directly with the expression of PPARG, ADIPOQ, and GLUT4 in human visceral and sc adipose tissue. Thus, PRL, acting through its cognate receptors, promotes healthy adipose tissue function and systemic insulin sensitivity. Increasing the levels of PRL in the circulation may have therapeutic potential against obesity-induced metabolic diseases.

Insulin-like growth factor 1 (IGF-1) can provide long-term neurotrophic support by activation of Akt, inhibition of FoxO nuclear localization and suppression of Bim gene transcription in multiple neuronal systems. However, MEK/ERK activation can also promote neuron survival through phosphorylation of BimEL. We explored the contribution of the PI3K/Akt/FoxO and MEK/ERK/BimEL pathways in IGF-1 stimulated survival after serum deprivation (SD) of R28 cells differentiated to model retinal neurons. IGF-1 caused rapid activation of Akt leading to FoxO1/3-T32/T24 phosphorylation, and prevented FoxO1/3 nuclear translocation and Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Overexpression of FoxO1 increased Bim mRNA expression and amplified the apoptotic response to SD without shifting the serum response curve. Inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1, while inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective during SD, while co-silencing of FoxO1 and Fox03 expression had little effect. Thus, the PI3K/Akt/FoxO pathway was not essential for protection from SD-induced apoptosis by IGF-1 in R28 cells. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. These studies demonstrate the requirement of the MEK/ERK pathway in a model of retinal neuron cell survival and highlight the cell specificity for IGF-1 signaling in this response.

The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca2+ rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr−/−) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

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, αvβ5 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.

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.

Vasoinhibins are prolactin fragments present in the retina, where they have been shown to prevent the hypervasopermeability associated with diabetes. Enhanced bradykinin (BK) production contributes to the increased transport through the blood-retina barrier (BRB) in diabetes. Here, we studied if vasoinhibins regulate BRB permeability by targeting the vascular endothelium and retinal pigment epithelium (RPE) components of this barrier. Intravitreal injection of BK in male rats increased BRB permeability. Vasoinhibins prevented this effect, as did the B2 receptor antagonist Hoe-140. BK induced a transient decrease in mouse retinal and brain capillary endothelial monolayer resistance that was blocked by vasoinhibins. Both vasoinhibins and the nitric oxide (NO) synthase inhibitor L-NAME, but not the antioxidant N-acetyl cysteine (NAC), blocked the transient decrease in bovine umbilical vein endothelial cell (BUVEC) monolayer resistance induced by BK; this block was reversed by the NO donor DETANONOate. Vasoinhibins also prevented the BK-induced actin cytoskeleton redistribution, as did L-NAME. BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC. DETANONOate reverted the blocking effect of vasoinhibins. Similar to BK, the radical initiator Luperox induced a reduction in ARPE-19 cell monolayer resistance, which was prevented by vasoinhibins. These effects on RPE resistance coincided with actin cytoskeleton redistribution. Intravitreal injection of vasoinhibins reduced the levels of reactive oxygen species (ROS) in retinas of streptozotocin-induced diabetic rats, particularly in the RPE and capillary-containing layers. Thus, vasoinhibins reduce BRB permeability by targeting both its main inner and outer components through NO- and ROS-dependent pathways, offering potential treatment strategies against diabetic retinopathies.