To develop an innovative preclinical full-thickness macular hole (FTMH) model using porcine eyes and to evaluate the feasibility of decellularized human corneal lenticule (hCL) as a novel bio-graft for macular hole closure. Twenty-five eyes (n = 25) were obtained from pigs euthanized for food consumption and transported to the laboratory within 3 h. Surgical procedures were performed by the same experienced vitreoretinal surgeon using a Leica Proveo microscope (Proveo 8, Leica Microsystems Inc.) with integrated intraoperative optical coherence tomography (iOCT) technology. After core vitrectomy and posterior vitreous detachment, FTMH was induced using diathermy and a vitrectomy probe. hCLs (thickness 100–120 µm), collected from refractive surgery patients, were decellularized and introduced into the vitreous cavity. With the aid of a viscoelastic agent for stability, hCLs were positioned into the FTMH using microsurgical forceps. Placement and integration were evaluated by iOCT and immunofluorescence (IF) analysis. OCT images and IF analysis confirmed the successful creation of FTMHs ex vivo in porcine eyes, demonstrating the feasibility of hole induction in an ex vivo model. Preliminary OCT and IF analyses showed the successful implantation of decellularized hCLs at the FTMH site, which were correctly positioned in the subretinal space between the choroid and the retinal pigment epithelium. Our preliminary results suggest that decellularized hCLs can be successfully implanted to close FTMHs in our preclinical ex vivo model of study, paving the way for future research into bioengineered hCLs for therapeutic purposes. Video available for this article.

Human stromal corneal lenticules (hSCLs) extracted during keratorefractive lenticule extraction (KLEx) refractive surgery, including Small incision lenticule extraction (SMILE) procedure, are often discarded as medical waste, despite their potential for reimplantation in various ocular therapeutic applications. These include corneal thickness restoration in keratoconus, scleral patch grafts, and other ophthalmic procedures. Ensuring the safety and structural integrity of these lenticules for clinical use depends on proper preservation; however, a consensus on the most effective preservation method has yet to be reached. Hence, this study aimed to establish a comprehensive preservation protocol of SMILE-hSCLs, evaluating both pre-storage transportation media and long-term storage methods. In particular, we tested two pre-storage transport media, hyaluronic acid (HA) and Coldix, a dextran-containing MEM medium commonly used in our eye bank for corneal hypothermic storage, and three long-term (two weeks) preservation techniques represented by silica gel dehydration, cryopreservation in DMSO and glycerol. Our results demonstrated that hSCLs transported in either HA or Coldix for 48 h retained transparency, size, morphology, thickness and biological properties comparable to fresh tissues. However, hSCLs transported in HA exhibited superior overall collagen structural integrity. While, among the three tested long-term storage methods, both dehydration and cryopreservation in glycerol ensured effective preservation of corneal tissue. In summary, our findings identified HA as an effective pre-storage transport medium and demonstrated that both silica gel dehydration and glycerol-based cryopreservation are suitable strategies for the long-term preservation of SMILE-derived hSCLs. Although cryopreservation ensures good tissue preservation, the need for specialized equipment may pose a barrier to implementation in certain settings. In contrast, dehydration might offer a practical, low-cost solution ideal for establishing off-the-shelf hSCL biobanks, particularly in resource-limited settings. Collectively, these results lay the groundwork for a standardized and scalable preservation protocol to support future clinical use of KLEx-derived lenticules.

IntroductionSmall incision lenticule extraction (SMILE)-derived lenticules have been repurposed as biocompatible scaffolds to incorporate and release therapeutic substances for ocular therapeutics. We aim to investigate the in vitro and in vivo release profiles of recombinant human nerve growth factor (rhNGF) from bioengineered human stromal lenticules prepared with microparticles incorporated with rhNGF (rhNGF-MPs) for up to 1 and 4 weeks, respectively.MethodsUpon bioengineered lenticule implantation, slit lamp, Anterior Segment Optical Coherence Tomography, and In Vivo Confocal Microscopy were performed to assess corneal biocompatibility, central corneal thickness (CCT), corneal nerve -fiber density (CNFD), -branch density, and -fiber length. Rabbit cornea, tears and aqueous humour were collected to quantify rhNGF release in vivo.ResultsA rapid in vitro release of rhNGF was detected until day 2, and with sustained release over 7 days. The pattern remains comparable even with the presence of antibiotic-antimycotic, trypan blue or fluorescein. Throughout the in vivo follow-up, no signs of corneal haze, edema, infiltration, or pathological increase in CCT were observed. A significant increase in CNFD (p = 0.001) at week 4 was reported in rhNGF-MPs than in Blank-MPs group. Finally, significantly higher NGF content in rabbit cornea and tear was found in rhNGF-MPs group compared to the endogenous NC group (p = 0.035 and p = 0.043, respectively).DiscussionBioengineered lenticules exhibit sustained rhNGF release for at least 7 days in vitro and up to 1 month in vivo. These results, together with absence of adverse effects, and significant increase in CNFD at 4 weeks after lenticule implantation suggest its promising potential for clinical use.

Introduction:Diabetes mellitus is a severe metabolic disorder strongly linked to vascular complications driven by endothelial dysfunction, chronic inflammation, and oxidative stress. Novel strategies to mitigate endothelial activation are urgently needed. In this context, phenolic compounds derived from olive leaves, a byproduct of olive oil production, have shown promising potential in counteracting diabetes- associated endothelial inflammation. This study investigates the potential anti-inflammatory effect of polyphenol-rich extracts derived from two olive leaves Spanish monocultivars, Picual and Changlot Real, in human umbilical vein endothelial cells from healthy pregnancies (C-HUVEC) and gestational diabetes (GD-HUVEC), which serve as a relevant in vitro model of hyperglycemia-induced endothelial dysfunction. Methods:Olive leaf extracts were characterized by HPLC-ESI-TOF-MS. C-HUVEC and GD-HUVEC were treated with the extracts, and pro-inflammatory markers expression (NF-kB p65, MCP-1, and VCAM-1), NF-kB p65 phosphorylation, and monocyte adhesion were assessed under basal and TNFα-stimulated conditions using RT-PCR, flow cytometry, and adhesion assays. Results:Both Picual and Changlot Real extracts showed no cytotoxicity at concentrations up to 50 mg/mL. Treatment with 10 mg/mL of both extracts significantly reduced NF-kB p65 and MCP-1 gene expression, as well as NF-kB p65 phosphorylation, particularly in GD-HUVEC. VCAM-1 protein expression and TNFα-induced monocyte adhesion were also significantly decreased following extract treatment. Notably, Changlot Real exhibited a broader anti-inflammatory effect across both cell types, while Picual exerted a more selective effect in GD-HUVEC. Discussion:These findings support the anti-inflammatory activity of olive leaf polyphenols and highlight the potential of Changlot Real and Picual extracts in mitigating endothelial dysfunction associated with diabetes. By modulating the NF-kB-VCAM-1 axis, these compounds may attenuate endothelial activation, warranting further investigation into their possible role in the prevention or mitigation of diabetes-related vascular complications.

ReLEx (Refractive Lenticule Extraction) Small Incision Lenticule Extraction (SMILE), the second generation of ReLEx Femtosecond Lenticule Extraction (FLEx), is a minimally invasive, flapless procedure designed to treat refractive errors such as myopia, hyperopia, presbyopia, and astigmatism. This review aims to provide a comprehensive overview of the methods for preserving SMILE-derived lenticules and discusses their potential future applications. A narrative literature review was conducted using PubMed, Scopus, and Web of Science databases, focusing on articles published up to January 2024 and available in English. The authors also evaluated the reference lists of the collected papers to identify any additional relevant research. No standardized protocols currently exist for the storage or clinical application of SMILE-derived lenticules. However, these lenticules present a promising resource for therapeutic uses, particularly in addressing the shortage of donor corneal tissues. Their potential applications include inlay and overlay additive keratoplasty, as well as other ocular surface applications. Further research is needed to establish reliable protocols for their preservation and clinical use. SMILE-derived lenticules offer significant potential as an alternative to donor corneal tissues. Standardizing their storage and application methods could enhance their use in clinical settings.

IntroductionDiabetic retinopathy (DR) is a microvascular complication of diabetes in which neurodegeneration has been recently identified as a driving force. In the last years, mesenchymal stromal cells (MSCs) and neurotrophins like Nerve Growth Factor (NGF), have garnered significant attention as innovative therapeutic approaches targeting DR-associated neurodegeneration. However, delivering neurotrophic factors directly in the eye remains a challenge. Hence, this study evaluated the effects of MSCs from human amniotic fluids (hAFSCs) and recombinant human NGF (rhNGF) delivered by human corneal lenticule (hCL) on a high glucose (HG) induced ex vivo model simulating the molecular mechanisms driving DR.MethodsPorcine neuroretinal explants exposed to HG (25 mM for four days) were used to mimic DR ex vivo. hCLs collected from donors undergoing refractive surgery were decellularized using 0.1% sodium dodecyl sulfate and then bioengineered with hAFSCs, microparticles loaded with rhNGF (rhNGF-PLGA-MPs), or both simultaneously. Immunofluorescence (IF) and scanning electron microscopy (SEM) analyses were performed to confirm the hCLs bioengineering process. To assess the effects of hAFSCs and rhNGF, bioengineered hCLs were co-cultured with HG-treated neuroretinal explants and following four days RT-PCR and cytokine array experiments for inflammatory, oxidative, apoptotic, angiogenic and retinal cells markers were performed.ResultsData revealed that HG-treated neuroretinal explants exhibit a characteristic DR-phenotype, including increased level of NF-kB, NOS2, NRF2 GFAP, VEGFA, Bax/Bcl2 ratio and decreased expression of TUBB3 and Rho. Then, the feasibility to bioengineer decellularized hCLs with hAFSCs and rhNGF was demonstrated. Interestingly, co-culturing hAFSCs- and rhNGF- bioengineered hCLs with HG-treated neuroretinal explants for four days significantly reduced the expression of inflammatory, oxidative, apoptotic, angiogenic and increased retinal markers.ConclusionOverall, we found for the first time that hAFSCs and rhNGF were able to modulate the molecular mechanisms involved in DR and that bioengineered hCLs represents a promising ocular drug delivery system of hAFSCs and rhNGF for eye diseases treatment. In addition, results demonstrated that porcine neuroretinal explants treated with HG is a useful model to reproduce ex vivo the DR pathophysiology.

Aims/Purpose: Retinal neurodegenerative diseases (RND) are associated with a progressive degeneration and death of retinal cells. Recently, mesenchymal stem cells (MSCs) showed a protective role against retinal cells degeneration and apoptosis. However, due to the complexity of such diseases, there is the need to develop in vitro or ex vivo models which allow the study of RND pathogenesis and better understand the regenerative role of MSCs. Therefore, the aim of our study was to use porcine retinas to establish a possible ex vivo model of spontaneous neuroretinal degeneration and employ this model to evaluate the efficacy of human amniotic fluid stem cells (hAFSCs) on retinal cells undergoing damage. Methods: To this end, neuroretinal explants were obtained from pig eyes and placed over the porous membrane of a transwell plate. To induce a time‐dependent spontaneous retinal degeneration, explants were cultured for 24, 48, 72, 96 h, 5 and 8 days. The damage grade was assessed with qRT‐PCR and immunohistochemistry analyses for GFAP and TUNEL‐assay. Then, the explants were co‐cultured with hAFSCs up to 8 days to evaluate their effect on retinal cells death via propidium iodide (PI) staining. Results: Results demonstrated that the cultivation of retinal explants up to 8 days induced an increase in the gene expression of inflammatory (NF‐kB and TNF‐alpha), pro‐apoptotic (p21 and p53) and oxidative stress markers (NRF2). We also observed an increased expression of GFAP and CD11b, suggesting an activation of the microglia and up‐regulation of Müller cells. In addition, the cultivation time induced an increase in the number of apoptotic cells (TUNEL). However, interestingly, the cells death decreased after co‐cultivation with hAFSCs (PI staining). Conclusions: In conclusion, our data demonstrate that the prolonged cultivation of porcine ex vivo retina can induce a time‐dependent spontaneous degeneration able to simulate a model of RND. Of note, the co‐culture preliminary data suggest a protective effects of hAFSCs on retinal cells death.

Background and aims: Hyperglycaemia during gestational diabetes (GD) predisposes women and their offspring to later cardiometabolic disease. The hyperglycaemia-mediated epigenetic changes remain to be elucidated. Methyltransferase MLL1-induced trimethylation of histone 3 at lysine 4 (H3K4me3) activates inflammatory and oxidative phenotype. This epigenetic mark in GD women and its transmission to the offspring were investigated. Methods: Peripheral blood mononuclear cells (PBMC) were collected from GD and control (C) women and also from adolescents born to women of both groups. Endothelial human umbilical vein endothelial cells (HUVEC) and cord blood mononuclear cells (CBMC) were from umbilical cords. The NF-kappa Bp65 and NOX4 expressions were investigated by reverse transcription quantitative polymerase chain reaction and immunofluorescence (IF). MLL1 and H3K4me3 were investigated by immunoblotting and IF. H3K4me3 on NF-kappa Bp65 and NOX4 promoters was studied by chromatin immunoprecipitation. Superoxide anion generation was measured by electron spin resonance spectroscopy. Plasma cytokines were measured by enzyme-linked immunosorbent assay. To investigate the role of MLL1, HUVEC were exposed to inhibitor MM102 or siRNA transfection. Results: PBMC, CBMC, and HUVEC showed an increase of NF-kappa Bp65, IL-6, ICAM-1, MCP-1, and VCAM-1 mRNAs. These findings were associated with H3K4me3 enrichment in the promoter of NF-kappa Bp65. Elevated H3K4me3 and cytokine levels were observed in GD adolescents. MLL1 drives H3K4me3 not only on NF-kB p65, but also on NOX4 promoter. Inhibition of MLL1 blunted NF-kappa Bp65 and NOX4 by modulating inflammatory and oxidative phenotype. Conclusions: Such proof-of-concept study shows persistence of MLL1-dependent H3K4me3 in offspring born to GD women, suggesting an epigenetic-driven transmission of maternal phenotype. These findings may pave the way for pharmacological reprogramming of adverse histone modifications to mitigate abnormal phenotypes underlying early ASCVD.

BACKGROUND:We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress. METHODS:II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue. RESULTS:Our protocol resulted in a yield of 6.31 ± 0.98 × 109 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H2O2 stress and TGFβ stimulation showed improved survival with a remarkable decrease in the onset of fibrosis. CONCLUSIONS:Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p-Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho-rhino-phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2-related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7-treated cells. Moreover, Fr7 counteracted H2O2-triggered cell damage, preventing increases in the pro-inflammatory and anti-chondrogenic microRNA (miR), miR-221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Type 2 diabetes (T2D) is a worldwide health problem and cardiovascular disease (CVD) is a leading cause of morbidity and mortality in T2D patients, making the prevention of CVD onset a major priority. It is therefore crucial to optimize diagnosis and treatment to reduce this burden. Endothelial dysfunction is one of the most important prognostic factors for CVD progression, thus novel approaches to identify the early phase of endothelial dysfunction may lead to specific preventive measures to reduce the occurrence of CVD. Nowadays, multiomics approaches have provided unprecedented opportunities to stratify T2D patients into endotypes, improve therapeutic treatment and outcome and amend the survival prediction. Among omics strategies, epigenetics and metabolomics are gaining increasing interest. Recently, a dynamic correlation between metabolic pathways and gene expression through chromatin remodeling, such as DNA methylation, has emerged, indicating new perspectives on the regulatory networks impacting cellular processes. Thus, a better understanding of epigenetic-metabolite relationships can provide insight into the physiological processes altered early in the endothelium that ultimately head to disease development. Here, recent studies on epigenetics and metabolomics related to CVD prevention potentially useful to identify disease biomarkers, as well as new therapies hopefully targeting the early phase of endothelial dysfunction are highlighted.

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo‐angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C‐HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD‐HUVECs), leading to a permanent pro‐inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti‐inflammatory and pro‐angiogenic properties of OA are still under investigation. We tested OA on C‐ and GD‐HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA‐pre‐treated C‐ and GD‐HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD‐HUVEC endothelium under OA pre‐treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD‐HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C‐ and GD‐HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.

Multiomics Multiomics approaches have provided unprecedented opportunities to stratify type 2 diabetes patients into endotypes and improve therapeutic treatments. A dynamic correlation between metabolic pathways and gene expression through chromatin remodeling has emerged, indicating new perspectives on the regulatory networks impacting cellular processes. In article number 2300172, Caterina Pipino and co-workers discuss the state of the art in this field.

Nowadays, ocular drug delivery still remains a challenge, since the conventional dosage forms used for anterior and posterior ocular disease treatments, such as topical, systemic, and intraocular administration methods, present important limitations mainly related to the anatomical complexity of the eye. In particular, the blood–ocular barrier along with the corneal barrier, ocular surface, and lacrimal fluid secretion reduce the availability of the administered active compounds and their efficacy. These limitations have increased the need to develop safe and effective ocular delivery systems able to sustain the drug release in the interested ocular segment over time. In the last few years, thanks to the innovations in the materials and technologies employed, different ocular drug delivery systems have been developed. Therefore, this review aims to summarize the synthetic and natural drug-loaded ocular inserts, contacts, and intraocular lenses that have been recently developed, emphasizing the characteristics that make them promising for future ocular clinical applications.

Extracellular vesicles (EVs) are a class of circulating entities that are involved in intercellular crosstalk mechanisms, participating in homeostasis maintenance, and diseases. Celiac disease is a gluten-triggered immune-mediated disorder, characterized by the inflammatory insult of the enteric mucosa following local lymphocytic infiltration, resulting in villous atrophy. The goal of this research was the assessment and characterization of circulating EVs in celiac disease patients, as well as in patients already on an adequate gluten-free regimen (GFD). For this purpose, a novel and validated technique based on polychromatic flow cytometry that allowed the identification and enumeration of different EV sub-phenotypes was applied. The analysis evidenced that the total, annexin V+, leukocyte (CD45+), and platelet (CD41a+) EV counts were significantly higher in both newly diagnosed celiac disease patients and patients under GFD compared with the healthy controls. Endothelial-derived (CD31+) and epithelial-derived (EpCAM+) EV counts were significantly lower in subjects under gluten exclusion than in celiac disease patients, although EpCAM+ EVs maintained higher counts than healthy subjects. The numbers of EpCAM+ EVs were a statistically significant predictor of intraepithelial leukocytes (IEL). These data demonstrate that EVs could represent novel and potentially powerful disease-specific biomarkers in the context of celiac disease.