Gonzalo Martínez de la Escalera

Vasoinhibins: endogenous regulators of angiogenesis and vascular function

Vasoinhibins are a family of peptides derived from prolactin, growth hormone and placental lactogen that act on endothelial cells to suppress vasodilation and angiogenesis and to promote apoptosis-mediated vascular regression. Some of the pathways by which vasoinhibins act have now been defined, and recent developments indicate that endogenous vasoinhibins exert tonic and essential actions on blood vessel growth, dilation and regression in vivo. By studying the pathways that can generate vasoinhibins, and the nature of their receptors and key biological mediators, it should be possible to clarify the role of vasoinhibins in controlling vascular function in health and disease.

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8>MMP-13 >MMP-3>MMP-1=MMP-2>MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.

Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopathy in rats via protein phosphatase 2A–dependent eNOS inactivation

Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies.

Elevated vasoinhibins may contribute to endothelial cell dysfunction and low birth weight in preeclampsia

Vasoconstriction and defective placental angiogenesis are key factors in the etiology of preeclampsia. Prolactin levels are elevated in maternal blood throughout pregnancy and the human decidua produces prolactin that is transported to the amniotic fluid. Prolactin is cleaved to yield vasoinhibins, a family of peptides that inhibit angiogenesis and nitric oxide-dependent vasodilation. Here, we conducted a case–control study to measure vasoinhibins in serum, urine, and amniotic fluid obtained from women with severe preeclampsia. We show that all three biological fluids contained significantly higher levels of vasoinhibins in preeclamptic women than in normal pregnant women. Amniotic fluid from preeclamptic women, but not from normal women, inhibited vascular endothelial growth factor-induced endothelial cell proliferation and nitric oxide synthase activity in cultured endothelial cells, and these actions were reversed by antibodies able to neutralize the effects of vasoinhibins. Furthermore, amniotic fluid does not appear to contain neutral prolactin-cleaving proteases, suggesting that vasoinhibins in amniotic fluid are derived from prolactin cleaved within the placenta. Also, cathepsin-D in placental trophoblasts cleaved prolactin to vasoinhibins, and its activity was higher in placental trophoblasts from preeclamptic women than from normal women. Importantly, birth weight of infants in preeclampsia inversely correlated with the extent to which the corresponding AF inhibited endothelial cell proliferation and with its concentration of prolactin+vasoinhibins. These data demonstrate that vasoinhibins are increased in the circulation, urine, and amniotic fluid of preeclamptic women and suggest that these peptides contribute to the endothelial cell dysfunction and compromised birth weight that characterize this disease.

17-Beta-Estradiol Directly Regulates the Expression of Adrenergic Receptors and Kisspeptin/GPR54 System in GT1-7 GnRH Neurons

Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17β-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) α1A-, α1B-, α1D-, α2A-, α2C-, and β1-AR, and showed for the first time that mRNAs for α2B-, β2- and β3-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17β-estradiol, α1B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while α1B- and α1D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1–10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.

Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells

Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1β. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium.

Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.

HIGH LEVELS OF SERUM PROLACTIN PROTECT AGAINST DIABETIC RETINOPATHY BY INCREASING OCULAR VASOINHIBINS

OBJECTIVE Increased retinal vasopermeability (RVP) occurs early in diabetes and is crucial for the development of sight-threatening proliferative diabetic retinopathy (DR). The hormone prolactin (PRL) is proteolytically processed to vasoinhibins, a family of peptides that inhibit the excessive RVP related to DR. Here, we investigate the circulating levels of PRL in association with DR in men and test whether increased circulating PRL, by serving as a source of ocular vasoinhibins, can reduce the pathological RVP in diabetes. RESEARCH DESIGN AND METHODS Serum PRL was evaluated in 40 nondiabetic and 181 diabetic men at various stages of DR. Retinal vasoinhibins were measured in rats rendered hyperprolactinemic by placing two anterior pituitary grafts under the kidney capsule and in PRL receptor–null mice. RVP was determined in hyperprolactinemic rats subjected to the intraocular injection of vascular endothelial growth factor (VEGF) or made diabetic with streptozotocin. RESULTS The circulating levels of PRL increased in diabetes and were higher in diabetic patients without retinopathy than in those with proliferative DR. In rodents, hyperprolactinemia led to vasoinhibin accumulation within the retina; genetic deletion of the PRL receptor prevented this effect, indicating receptor-mediated incorporation of systemic PRL into the eye. Hyperprolactinemia reduced both VEGF-induced and diabetes-induced increase of RVP. This reduction was blocked by bromocriptine, an inhibitor of pituitary PRL secretion, which lowers the levels of circulating PRL and retinal vasoinhibins. CONCLUSIONS Circulating PRL influences the progression of DR after its intraocular conversion to vasoinhibins. Inducing hyperprolactinemia may represent a novel therapy against DR.

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.

Prolactin promotes cartilage survival and attenuates inflammation in inflammatory arthritis

Chondrocytes are the only cells in cartilage, and their death by apoptosis contributes to cartilage loss in inflammatory joint diseases, such as rheumatoid arthritis (RA). A putative therapeutic intervention for RA is the inhibition of apoptosis-mediated cartilage degradation. The hormone prolactin (PRL) frequently increases in the circulation of patients with RA, but the role of hyperprolactinemia in disease activity is unclear. Here, we demonstrate that PRL inhibits the apoptosis of cultured chondrocytes in response to a mixture of proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) by preventing the induction of p53 and decreasing the BAX/BCL-2 ratio through a NO-independent, JAK2/STAT3-dependent pathway. Local treatment with PRL or increasing PRL circulating levels also prevented chondrocyte apoptosis evoked by injecting cytokines into the knee joints of rats, whereas the proapoptotic effect of cytokines was enhanced in PRL receptor-null (Prlr(-/-)) mice. Moreover, eliciting hyperprolactinemia in rats before or after inducing the adjuvant model of inflammatory arthritis reduced chondrocyte apoptosis, proinflammatory cytokine expression, pannus formation, bone erosion, joint swelling, and pain. These results reveal the protective effect of PRL against inflammation-induced chondrocyte apoptosis and the therapeutic potential of hyperprolactinemia to reduce permanent joint damage and inflammation in RA.