Celina García

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.

Vasoinhibins: a family of N-terminal prolactin fragments that inhibit angiogenesis and vascular function.

Antiangiogenic molecules derived from prolactin (PRL) are not a single entity, but rather a family of peptides with different molecular masses, all containing the N-terminal region of PRL. Cleavage of PRL by cathepsin-D or by matrix metalloproteases generates N-terminal fragments that act on endothelial cells to suppress vasodilation and angiogenesis and promote vascular regression. N-terminal PRL fragments have been identified in cartilage and retina, where angiogenesis is highly restricted. In vivo experiments demonstrate that these PRL fragments exert a tonic and essential suppression of retinal blood vessel growth and dilation. Similar PRL fragments have been detected in the pituitary gland, a highly vascularized organ where the control of vascular growth may differ from that in tissues where angiogenesis is highly restricted. We have previously proposed the name vasoinhibins to describe the collection of N-terminal PRL fragments having blood vessel-blocking activity, and here we discuss their promise as factors to control vascular function in health and disease.

Vasoinhibins: novel inhibitors of ocular angiogenesis

Disruption of the quiescent state of blood vessels in the retina leads to aberrant vasopermeability and angiogenesis, the major causes of vision loss in diabetic retinopathy. Prolactin is expressed throughout the retina, where it is proteolytically cleaved to vasoinhibins, a family of peptides (including the 16-kDa fragment of prolactin) with potent antiangiogenic, vasoconstrictive, and antivasopermeability actions. Ocular vasoinhibins act directly on endothelial cells to block blood vessel growth and dilation and to promote apoptosis-mediated vascular regression. Also, vasoinhibins prevent retinal angiogenesis and vasopermeability associated with diabetic retinopathy, and inactivation of endothelial nitric oxide synthase via protein phosphatase 2A is among the various mechanisms mediating their actions. Here, we discuss the potential role of vasoinhibins both in the maintenance of normal retinal vasculature and in the cause and prevention of diabetic retinopathy and other vasoproliferative retinopathies.

Prolactin in Ovarian Follicular Fluid Stimulates Endothelial Cell Proliferation

Angiogenesis is essential for the growth and maturation of the ovarian follicle and its transition into the corpus luteum. In addition to the main proangiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), follicular fluid (FF) contains the hormone prolactin (PRL), which is known to promote angiogenesis in vivo. Here, we show that FF from large follicles, which contains twice the PRL level of FF from small follicles, stimulates endothelial cell proliferation to a greater extent than the latter, and that immunoneutralization of PRL prevents FF from stimulating endothelial cell proliferation. Notably, the FF increases the expression of the short and long PRL receptor isoforms in endothelial cells, and a purified PRL standard stimulates endothelial cell proliferation but only after the cells have been pretreated with FF. However, purified PRL activates the JAK2/STAT3 pathway in endothelial cells in the absence of pretreatment with FF. In summary, PRL present in the FF stimulates the proliferation of endothelial cells. This effect likely involves the upregulation of the short and long PRL receptor isoforms and is independent of PRL-induced JAK2/STAT3 signaling.

Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK). Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS), as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i) upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC) channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.