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Diabetic retinopathy is a secondary microvascular complication of diabetes mellitus. This disease progresses from two stages, non-proliferative and proliferative diabetic retinopathy, the latter characterized by retinal abnormal angiogenesis. Pharmacological management of retinal angiogenesis employs expensive and invasive intravitreal injections of biologic drugs (anti-vascular endothelial growth factor agents). To search small molecules able to act as anti-angiogenic agents, we focused our study on axitinib, which is a tyrosine kinase inhibitor and represents the second line treatment for renal cell carcinoma. Axitinib is an inhibitor of vascular endothelial growth factor receptors, and among the others tyrosine kinase inhibitors (sunitinib and sorafenib) is the most selective towards vascular endothelial growth factor receptors 1 and 2. Besides the well-known anti-angiogenic and immune-modulatory functions, we hereby explored the polypharmacological profile of axitinib, through a bioinformatic/molecular modeling approach and in vitro models of diabetic retinopathy. We showed the anti-angiogenic activity of axitinib in two different in vitro models of diabetic retinopathy, by challenging retinal endothelial cells with high glucose concentration (fluctuating and non-fluctuating). We found that axitinib, along with inhibition of vascular endothelial growth factor receptors 1 (1.82 ± 0.10; 0.54 ± 0.13, phosphorylated protein levels in fluctuating high glucose vs. axitinib 1 µM, respectively) and vascular endothelial growth factor receptors 2 (2.38 ± 0.21; 0.98 ± 0.20, phosphorylated protein levels in fluctuating high glucose vs. axitinib 1 µM, respectively), was able to significantly reduce (p < 0.05) the expression of Nrf2 (1.43 ± 0.04; 0.85 ± 0.01, protein levels in fluctuating high glucose vs. axitinib 1 µM, respectively) in retinal endothelial cells exposed to high glucose, through predicted Keap1 interaction and activation of melanocortin receptor 1. Furthermore, axitinib treatment significantly (p < 0.05) decreased reactive oxygen species production (0.90 ± 0.10; 0.44 ± 0.06, fluorescence units in high glucose vs. axitinib 1 µM, respectively) and inhibited ERK pathway (1.64 ± 0.09; 0.73 ± 0.06, phosphorylated protein levels in fluctuating high glucose vs. axitinib 1 µM, respectively) in HRECs exposed to high glucose. The obtained results about the emerging polypharmacological profile support the hypothesis that axitinib could be a valid candidate to handle diabetic retinopathy, with ancillary mechanisms of action.

We have shown previously that glycogen synthase kinase-3[beta] (GSK-3[beta]), cyclin-dependent kinase 5, and c-Jun N[H.sub.2]-terminal kinase become overactivated and hyperphosphorylate [tau] in heat-shocked female rats. This hyperphosphorylation of [tau] is estrogen-independent, prevented by androgens, and similar to Alzheimer's disease. In this study, ovariectomized (OVX) Sprague-Dawley rats (n = 75) received daily injections of 10 [micro]g of 17[beta]-estradiol benzoate (EB), or 250 [micro]g of testosterone propionate (TP), or both EB and TP, or sesame oil (SO) vehicle for 4-6 weeks. In kinase assays of forebrain homogenates, overactivation of GSK-3[beta] at 0-6 h after heat shock toward human recombinant [tau], bovine [tau], and phosphoglycogen synthase peptide 2 was prevented in OVX + TP and OVX + (EB + TP) but not in sham-OVX + SO, OVX + SO, and OVX + EB. Abs against inactive (p[Ser.sup.9]) and activity-enhanced (p[Tyr.sup.216]) GSK-3[beta] showed marked increase of p[Ser.sup.9]- and decrease of p[Tyr.sup.216]- GSK-3[beta] in both OVX + TP and OVX + (EB + TP) but not in sham-OVX + SO, OVX + SO, and OVX + EB. EB enhanced the overactivation of cyclin-dependent kinase 5. The activity of c-Jun N[H.sub.2]-terminal kinase was gonadal hormone-independent. The serum concentrations of testosterone and 17[beta]-estradiol were 2.53 ng/ml and 201 pg/ml in OVX + TP and OVX + EB, respectively. These findings demonstrate that testosterone prevents the hyperphosphorylation of [tau] by inhibiting the heat shock-induced overactivation of GSK-3[beta] and suggest that androgens given to aging men or, in combination with estrogens, to postmenopausal women could prevent or delay Alzheimer's disease.

We have previously shown, by using the phosphate-dependent anti-[Tau] antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of [Tau] followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague-Dawley rats the activities of extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun [NH.sub.2]-terminal kinase (JNK), glycogen synthase kinase-3[Beta] (GSK-3[Beta]), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) at 0 (n = 5), 3 (n = 4), 6 (n = 5), and 12 (n = 5) h after heat shock and in non-heat-shocked controls (n = 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3[Beta], which showed increased activity. At 3-6 h, the activities of ERK1/2, JNK, Cdk5, and GSK-3[Beta] toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3[Beta] but not ERK1/2 were overactivated toward purified bovine [Tau]. At 3-6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either [Tau] or selective substrates. All of eight anti-[Tau] antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3-6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1/2, JNK, and GSK-3[Beta] confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because [Tau] is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3[Beta], and Cdk5 may play a role in its pathogenesis.

Analyzes the activities of different types of protein kinases in forebrain homogenates from female rats after heat shock. Increase activation and inhibition of kinases; Description of a proposed rat heat shock model; Possible role of the protein kinases in the pathogenesis of Alzheimer disease.