María-Celia Morales

Plasma rich in growth factors as a therapeutic agent for persistent corneal epithelial defects.

Objective: To evaluate the efficacy of topically applied autologous plasma rich in growth factors (PRGF) as a treatment for persistent epithelial defects (PEDs) of the cornea. Methods: A series of prospective noncomparative cases. Participants: Twenty eyes from 18 patients with PED with various underlying etiopathologies: neurogenic, iatrogenic, associated with burning or secondary to severe dry eye. Patients were treated with a PRGF eyedrop solution. Serial photographs of the cornea were taken until epithelialization was complete. We had previously characterized the levels of a panel of growth factors (platelet-derived growth factor, epithelial growth factor, vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor, and nerve growth factor) in the PRGF of 11 of these patients. The following variables were additionally recorded: (1) duration of PED before treatment, (2) previous treatments, (3) time for complete epithelialization, and (4) treatments required concomitantly with PRGF. Results: Epithelial defects healed in 17 of 20 cases (85%), with a mean therapeutic time of 10.9 weeks (range 2-39 weeks). Mean progression time before treatment was 26.7 weeks (range 2-104 weeks). Growth factor concentrations were platelet-derived growth factor 12645.9 ± 1690.0 pg/mL, epithelial growth factor 468.9 ± 97.6 pg/mL, vascular endothelial growth factor 204.5 ± 119.4 pg/mL, hepatocyte growth factor 149.5 ± 173.5 pg/mL, fibroblast growth factor 82.6 ± 95.9 pg/mL, and nerve growth factor 37.7 ± 18.6 pg/mL. Conclusion: PRGF, when applied as eyedrops, is a highly effective therapeutic agent for the treatment of a broad etiopathological spectrum of corneal PEDs.

In Vitro Effects of Three Blood Derivatives on Human Corneal Epithelial Cells

PURPOSE We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. METHODS The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. RESULTS We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. CONCLUSIONS PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

Efficacy of plasma rich in growth factors for the treatment of dry eye.

Purpose: To evaluate the efficacy of plasma rich in growth factors (PRGF) for the treatment of moderate/severe dry eye. Methods: PRGF treatment was administered to 16 patients who had moderate/severe dry eye diagnosed and who had not responded previously to other standard treatments. We quantified several growth factors present in the PRGF of each patient and obtained quantitative registers of the symptoms (modified score dry eye questionnaire), both before and after PRGF treatment. We also performed impression cytology to determine the degree of squamous metaplasia before and after PRGF treatment. Results: PRGF treatment was associated with a statistically significant improvement in score dry eye questionnaire values (P < 0.001). Results from impression cytology corroborated this improvement, but the reduction in the degree of squamous metaplasia was not statistically significant. In 75% of patients treated with PRGF, no further treatments were required, whereas in the remaining 25% other ocular treatments could be reduced. Conclusions: PRGF led to symptom improvement in patients with moderate/severe dry eye. Surprisingly, the symptoms recorded in the dry eye questionnaire do not always agree with the degree of squamous metaplasia measured by impression cytology.

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.

Corneal Wound Healing Promoted by 3 Blood Derivatives: An In Vitro and In Vivo Comparative Study

Purpose: The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]. Methods: Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment. Results: In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment. Conclusions: All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.