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.

Sulpiride-induced hyperprolactinaemia increases retinal vasoinhibin and protects against diabetic retinopathy in rats.

Excessive vasopermeability and angiogenesis compromise vision in diabetic macular oedema (DME) and diabetic retinopathy (DR). Vasoinhibin is a fragment of the hormone prolactin (PRL) that inhibits diabetes-induced retinal hypervasopermeability and ischaemia-induced retinal angiogenesis in rodents. Hyperprolactinaemia generated by the dopamine D2 receptor antagonist, levosulpiride, is associated with higher levels of vasoinhibin in the vitreous of patients with DR, implying a beneficial outcome due to vasoinhibin-mediated inhibition of retinal vascular alterations. Here, we tested whether hyperprolactinaemia induced by racemic sulpiride increases intraocular vasoinhibin levels and inhibits retinal hypervasopermeability in diabetic rats. Diabetes was generated with streptozotocin and, 4 weeks later, rats were treated for 2 weeks with sulpiride or osmotic minipumps delivering PRL. ELISA, Western blot, and Evans blue assay were used to evaluate serum PRL, retinal vasoinhibin, and retinal vasopermeability, respectively. Hyperprolactinaemia in response to sulpiride or exogenous PRL was associated with increased levels of vasoinhibin in the retina and reduced retinal hypervasopermeability. Furthermore, sulpiride decreased retinal haemorrhages in response to the intravitreal administration of vascular endothelial growth factor (VEGF). Neither sulpiride nor exogenous PRL modified blood glucose levels or bodyweight. We conclude that sulpiride-induced hyperprolactinaemia inhibits the diabetes- and VEGF-mediated increase in retinal vasopermeability by promoting the intraocular conversion of endogenous PRL to vasoinhibin. These findings support the therapeutic potential of sulpiride and its levorotatory enantiomer, levosulpiride, against DME and DR.

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 (2 µg g-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.