Diego Guerrieri

Corneal alkali burns represent one of the most severe forms of ocular surface injury and frequently result in persistent inflammation, corneal neovascularization, stromal remodeling, and permanent visual impairment. Current therapeutic approaches incompletely control the inflammatory mechanisms that sustain pathological angiogenesis and tissue disorganization. In this study, we evaluated the effects of a transglutaminase-binding fusion protein (FP) in a rat model of alkali-induced corneal injury. Following standardized alkali burns, animals were treated topically with FP, secretory leukocyte protease inhibitor (SLPI), or Buffer. Corneal epithelial healing, opacity, and neovascularization were assessed clinically and by digital image-based quantification, while histological and immunofluorescence analyses were used to evaluate stromal organization and vascular invasion. Molecular mechanisms were investigated by RT-qPCR and Western blot analysis of key inflammatory, angiogenic, and signaling mediators. FP treatment significantly accelerated corneal re-epithelialization, reduced corneal opacity, and markedly attenuated corneal neovascularization compared to SLPI and Buffer controls. These effects were associated with coordinated downregulation of pro-inflammatory cytokines and angiogenic mediators, including TNF-α, IL-17, VEGF, and cPLA₂. Notably, FP suppressed transglutaminase 2 expression and induced early and sustained downregulation of NF-κB pathway components, identifying modulation of an upstream inflammatory pathway central to corneal angiogenesis and stromal remodeling. Collectively, these findings demonstrate that FP effectively limits inflammation-driven corneal neovascularization and tissue remodeling following alkali injury, supporting its potential as a disease-modifying therapeutic strategy for inflammatory ocular surface disorders.

Acute kidney injury (AKI) is characterized by rapid loss of excretory function and is the clinical manifestation of several disorders affecting the kidney. The aim of the present study was to investigate the mechanism of action of Secretory Leukocyte Proteinase Inhibitor (SLPI) that protects the kidneys form AKI. In vivo and in vitro experiments were performed to assess the effect of SLPI on kidney injury. Animal models of kidney injury was generated by 40 min obstruction of kidney artery and vein (ischemia-reperfusion injury model) or daily administration of 60 mg/kg/day of gentamicine for 5 day (gentamicin-associated AKI model). For in vitro assessment, human renal epithelium HK-2 cells were cultured under serum starvation conditions or with tacrolimus. The administration of SLPI (250 μg/kg, i.p.) reduced elevated plasma creatinine and blood urea nitrogen levels, tissue myeloperoxidase content, and acute tubular necrosis induced by kidney damage. Furthermore, SLPI treatment reduced CD86, CD68, CD14, CCL2, TNFα, and IL-10 transcripts in kidney biopsies. To further analyze a direct effect of SLPI on renal epithelial cells, HK-2 cells from human renal epithelium were cultured under serum starvation conditions or with tacrolimus. Both conditions induced apoptosis of HK-2 cells which was reduced when SLPI was present in the culture medium. Furthermore, SLPI favored the proliferation and migration of HK-2 cells. An analysis of the gene profiles of HK-2 cells treated with calcineurin inhibitors affected inflammatory and non-inflammatory pathways that were reversed by SLPI. Among them, SLPI down modulated the expression of CCL2, SLC5A3, and BECN1 but up-regulated the expression of TLR4, ATF4, ATF6, HSP90B, BBC3 SLC2A1, and TNFRSF10B. Overall, these results suggest that SLPI, in addition to its activity on immune cells, may directly target tubular epithelial cells of the kidney to mediate the nephroprotective activity in AKI.

To study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis. An alkali injury was produced with a filter paper of 3 mm with 1 N NaOH during 40 seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n = 12), Group 2, with 10 ul of SLPI (200 ug/ml; n = 12) and Group 3 was treated with buffer (10 ul; n = 12) topically administered four times a day for up to 7 days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24 hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence. Corneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18 hours and 24 hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7 days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC. The binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.