Prolactin and vasoinhibins: Endogenous players in diabetic retinopathy.

Diabetic retinopathy is a disease of the retinal microvasculature that develops as a complication of diabetes mellitus and constitutes a major cause of blindness in adults of all ages. Diabetic retinopathy is characterized by the loss of capillary cells leading to increased vasopermeability, ischemia, and hypoxia that trigger the excessive formation of new blood vessels in the retina. The influence of the pituitary gland in the pathophysiology of diabetic retinopathy was recognized nearly six decades ago, but the contribution of pituitary hormones to this disease remains unclear. Recent studies have shown that the pituitary hormone prolactin is proteolytically cleaved to vasoinhibins, a family of peptides with potent antivasopermeability, vasoconstrictive, and antiangiogenic actions that can protect the eye against the deleterious effects of the diabetic state. In this review, we summarize what is known about the changes in the circulating levels of prolactin and vasoinhibins during diabetes and diabetic retinopathy as well as the implications of these changes for the development and progression of the disease with particular attention to hyperprolactinemia in pregnancy and postpartum. We discuss the effects of prolactin and vasoinhibins that may impact diabetic retinopathy and suggest these hormones as important targets for therapeutic interventions.

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.

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.

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-1beta. 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.

Hypoxia inhibits expression of prolactin and secretion of cathepsin-D by the GH4C1 pituitary adenoma cell line.

Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas.