Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models

Experimental and clinical data suggest that pro-angiogenic, pro-inflammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms.Here we show, for the first time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ~40%. VEGF-dependent proliferation and migration were significantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100-250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efficacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 μg/eye) significantly reduced pathological vascularization with the efficacy similar to that of a standard treatment (anti-VEGF antibody, 1 μg/eye).Cumulatively, our results suggest that chronic exposure to VEGF upregulates leptin expression and function. As leptin can in turn activate VEGF, the increased abundance of both cytokines could amplify pro-angiogenic and pro-inflammatory environement in the eye. Thus, combined therapies targeting ObR and VEGF should be considered in the treatment of ocular diseases

Leptin mediates hyperglycemia-induced angiogenic effects in retinal endothelial cells

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXVII Ciclo, a.a. 2013-2014Hyperglycemia (HG)-activated cytokines and inflammatory factors have been implicated in ocular neovascularization and diabetic retinopathy (DR). However, the effects of HG on the expression and function of leptin in retinal cells have never been investigated. We found that in RF/6A retinal endothelial cells, chronic high glucose (30 mM/L) exposure significantly increased leptin mRNA expression and upregulated leptin protein and leptin receptor levels. Furthermore, HG potentiated RF/6A cell migration, chemokinesis, and angiogenic differentiation. These effects coincided with the activation of several intracellular pathways implicated in angiogenic and metabolic response, i.e., STAT3, ERK1/2, Akt, and AMPK, increased levels of glucose response protein and COX2 as well as modulation of the expression of PAI-2 and HIF-1α. All pro-angiogenic processes promoted by HG and several of HG-activated intracellular pathways were partially or totally blocked in the presence of the leptin receptor antagonist peptide. The results demonstrate for the first time that the leptin/leptin receptor axis is implicated in HG-induced biological effects in retinal endothelial cells. Thus, targeting leptin pathways might represent a novel avenue in the treatment of ocular neovascularizationUniversità della Calabri

Exploring leptin antagonism in ophthalmic cell models.

BACKGROUND:Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions. METHODS AND RESULTS:The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca. CONCLUSIONS:Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models

Effects of Allo-aca on leptin-dependent proliferation in RF/6A and BCE cells.

<p>RF/6A and BCE cells were synchronized in SFM and stimulated with 250 ng/mL leptin (L) in the presence or absence of 1-250 nM Allo-aca (Allo) for 24 h. The % increase/decrease (±SD) in cell number vs. untreated control (C=SFM) is shown. Asterisks indicate significant (p≤0.05) differences vs. leptin.</p

Effects of Allo-aca on leptin angiogenic activity in RF/6A and BCE cells.

<p>The assays were carried out as described in Materials and Methods. Untreated cells (C=SFM) and cells treated with 250 leptin (L) alone, L+ 1-100 nM Allo-aca (Allo), or 10-100 nM Allo-aca. The photographs represent ES formation under different treatments in RF/6A and BCE cells (central field of the well at 5x magnification is shown). The graph shows the number of ES (±SD) per visual field. Asterisks indicate significant (p≤0.05) differences vs. leptin.</p

Effects of leptin and Allo-aca on leptin protein expression in RF/6A and BCE cells.

<p>RF/6A and BCE cells were synchronized in SFM and stimulated with 250 ng/mL leptin (L) for 24 h in the presence or absence of 100 nM (for RF/6A) or 250 nM (for BCE) Allo-aca (Allo). The expression of leptin protein (green immunofluorescence) in treated and untreated cells was detected with specific Abs, as described in Materials and Methods. In control experiments, the primary Ab was omitted.</p

Effects of Allo-aca on intracellular leptin effects in RF/6A and BCE cells.

<p><b>A</b>. The effects of 10 and 100 nM Allo-aca (Allo) on acute intracellular signaling were tested in RF/6A and BCE cells stimulated with 250 ng/mL leptin (L) for 30 min. <b>B</b>. The long-term effects of 100 nM Allo-aca (Allo) on intracellular pathways were assessed in cells stimulated for 12 h (RF/6A) or 6 h (BCE) with 250 ng/mL leptin (L) and measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076437#pone-0076437-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076437#pone-0076437-g006" target="_blank">6</a>. The representative blots of at least 3 experiments are shown.</p

Long-term intracellular leptin effects in RF/6A and BCE cells.

<p>RF/6A and BCE cells were stimulated with 250 ng/mL leptin (L) for 6, 12, 24 h as described in Materials and Methods. The numbers under WB panels represent relative densitometry values (%) of phosphorylated and total proteins, with the value in SFM is taken as 100%. The representative blots of at least 3 experiments are shown.</p

Leptin effects on intracellular signaling in RF/6A and BCE cells.

<p>RF/6A and BCE cells were stimulated with 250 ng/mL leptin (L) for 15 and 30 min and the expression of phosphorylated (p) and total (Tot) proteins was assessed by WB and quantified as described in Materials and Methods. The levels of β-actin were assessed as control of loading. The numbers under WB panels represent relative densitometry values (%) of phosphorylated and total proteins, with the value in SFM is taken as 100%. The representative blots of at least 3 experiments are shown.</p