pEPito-driven PEDF Expression Ameliorates Diabetic Retinopathy Hallmarks

Diabetic retinopathy (DR) is one of the major complications of diabetes mellitus. It is characterized by retinal microvascular changes caused by chronic exposure to hyperglycemia, leading to low tissue oxygenation and ultimately to neovascularization. Laser photocoagulation and vitrectomy are the most efficient treatments for DR, but display severe side effects such as the destruction of the healthy retina. Another clinical approach uses antiangiogenic agents to prevent and delay progression of neovascularization, but these require recurrent local administrations that increase the possibility of retinal detachment, vitreous hemorrhage, and cataract formation. Studies in human diabetic retinas have revealed an imbalance between proangiogenic factors such as the vascular endothelial growth factor (VEGF) and antiangiogenic factors, such as pigment epithelial-derived factor (PEDF). This imbalance favors pathological angiogenesis contributing to DR, and can constitute a therapeutic target. Gene therapy was recently shown to be an adequate intervention for long-term treatment of several retinal pathologies. We have previously shown the newly engineered episomal vector pEPito to be able of sustained gene expression in the mouse retina. We here show that pEPito was able to overexpress PEDF for up to three months, both in in vitro cultures of human retinal pigment epithelial cells and in the retina of diabetic mice after a single subretinal injection. In vivo, in parallel with the increase in PEDF we observed a decrease in VEGF levels in injected compared with noninjected eyes and a significant effect on two hallmarks of DR: reduction of glucose transport (by glucose transporter GLUT1), and reduction of inflammation by decreased reactivity of microglia. Jointly, these results point to a significant therapeutic potential of gene therapy with pEPito-PEDF for the treatment of DR

Sustained gene expression in the retina by improved episomal vectors

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.Fundacao para a Ciencia e Tecnologia, Portugal [PEst/OE/EQB-LA 0023/2013, SFRH/BD/76873/2011, SFRH/BD/70318/2010, PTDC/SAU/BEB/098475/2008]; European Union [PIRG-GA-2009-249314

is there a link?

PMID:25076895Spinal cord infarction (SCI) is an uncommon but important cause of acute myelopathy. Nevertheless, contrary to cerebral stroke, the discussion about paradoxical embolism as a cause of cryptogenic SCI remains dubious. We describe the case of a 24-year-old woman who developed sudden-onset back pain followed by upper limb paralysis. T2-weighted MRI demonstrated hyperintense signal, extending from C5 to D1 with corresponding restricted diffusion on diffusion-weighted MRI and reduction of the apparent diffusion coefficient. Diagnostic workup, including lumbar puncture, showed no changes. Transcranial Doppler showed a right-to-left shunt with an uncountable number of microembolic signals after Valsalva maneuvers, and a patent foramen ovale (PFO) with an atrial septum aneurysm was identified. We discuss the paucity of evidence of right-to-left shunting in spinal diseases compared to cerebral events and the potential role of paradoxical embolism through PFO as a possible mechanism of SCI.publishersversionpublishe

Oxidative stress modulates the expression of VEGF isoforms in the diabetic retina

Funding: This work was supported by the Portuguese Foundation for Science and Technology (FCT) with individual grants to S. Simão (SFRH/ BPD/78404/2011), D. Bitoque (SFRH/BD/52424/2013), S. Calado (SFRH/BD/76873/2011), GA Silva (EXPL-BIM-MEC-1433-2013, PIRG05-GA-2009-249314–EyeSee). FCT Research Center Grant UID/ BIM/04773/2013 CBMR 1334.publishersversionpublishe

Human stem cells for cardiac disease modeling and preclinical and clinical applications—are we on the road to success?

Cardiovascular diseases (CVDs) are pointed out by the World Health Organization (WHO) as the leading cause of death, contributing to a significant and growing global health and economic burden. Despite advancements in clinical approaches, there is a critical need for innovative cardiovascular treatments to improve patient outcomes. Therapies based on adult stem cells (ASCs) and embryonic stem cells (ESCs) have emerged as promising strategies to regenerate damaged cardiac tissue and restore cardiac function. Moreover, the generation of human induced pluripotent stem cells (iPSCs) from somatic cells has opened new avenues for disease modeling, drug discovery, and regenerative medicine applications, with fewer ethical concerns than those associated with ESCs. Herein, we provide a state-of-the-art review on the application of human pluripotent stem cells in CVD research and clinics. We describe the types and sources of stem cells that have been tested in preclinical and clinical trials for the treatment of CVDs as well as the applications of pluripotent stem-cell-derived in vitro systems to mimic disease phenotypes. How human stem-cell-based in vitro systems can overcome the limitations of current toxicological studies is also discussed. Finally, the current state of clinical trials involving stem-cell-based approaches to treat CVDs are presented, and the strengths and weaknesses are critically discussed to assess whether researchers and clinicians are getting closer to success.B.M.S. was awarded with a Ph.D. fellowship (reference: 2022.13253.BDANA) by Fundação para a Ciência e Tecnologia (FCT). S.M.C is supported by a Stimulus of Scientific Employment, Individual Support (2020.01532.CEECIND) by FCT. J.B. is grateful to the FCT and the Comissão de Coordenação e Desenvolvimento Regional do Algarve (CCDR Algarve) for the project ALG-45-2020-41 and to Algarve Biomedical Center (ABC) for the award “Bolsa de Investigação Translacional—José Mariano Gago by ABC, 2022”. M.T.F. thanks the FCT for funding the project with the reference 2022.09209.PTDC.info:eu-repo/semantics/publishedVersio

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2 Akita

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2(Akita) mice that developed T1DM around 3-4 weeks after birth. Both Ins2(Akita) and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2(Akita) mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2(Akita) bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2(Akita). We suggest that Ins2(Akita) mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.F. R. Carvalho and S. M. Calado acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) through Ph.D. fellowships SFRH/BD/76429/2011 and SFRH/BD/76873/2011, respectively. This study was funded in part by CCMAR funding from European Regional Development Fund (ERDF) under COMPETE Program and through FCT under PEst-C/MAR/LA0015/2011 project and through UID/Multi/04326/2013 project. GA Silva was funded by (PIRG05-GA-2009-249314-EyeSee) and Research Center Grant UID/BIM/04773/2013 to CBMR

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.FCT: PTDC/BTM/ORG/28121/2017; PD/BD/52424/2013; SFRH/BD/76873/2011;PIRG-GA-2009-249314info:eu-repo/semantics/publishedVersio

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2 (Akita)

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.SFRH/BD/76873/2011/ SFRH/BD/76429/2011/ PEst-C/MAR/LA0015/2011info:eu-repo/semantics/publishedVersio

Interferon-alpha decreases cancer stem cell properties and modulates exosomes in malignant melanoma

Malignant melanoma (MM) can spread to other organs and is resistant in part due to the presence of cancer stem cell subpopulations (CSCs). While a controversial high dose of interferon-alpha (IFN-α) has been used to treat non-metastatic high-risk melanoma, it comes with undesirable side effects. In this study, we evaluated the effect of low and high doses of IFN-α on CSCs by analyzing ALDH activity, side population and specific surface markers in established and patient-derived primary cell lines. We also assessed the clonogenicity, migration and tumor initiation capacities of IFN-α treated CSCs. Additionally, we investigated genomic modulations related to stemness properties using microRNA sequencing and microarrays. The effect of IFN-α on CSCs-derived exosomes was also analyzed using NanoSight and liquid chromatography (LC-HRMS)-based metabolomic analysis, among others. Our results showed that even low doses of IFN-α reduced CSC formation and stemness properties, and led to a significant decrease in the ability to form tumors in mice xenotransplants. IFN-α also modulated the expression of genes and microRNAs involved in several cancer processes and metabolomics of released exosomes. Our work suggests the utility of low doses of interferon, combined with the analysis of metabolic biomarkers, as a potential clinical approach against the aggressiveness of CSCs in melanoma.This research was funded by the Ministerio de Ciencia, Innovación y Universidades (MICIU, projects noº MAT2015-62644.C2.2.R and RTI2018-101309-B-C2, FEDER Funds), by the Instituto de Salud Carlos III (PIE16-00045), by Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía and European Regional Development Fund (ERDF), ref. SOMM17/6109/UGR (UCE-PP2017-3), by Consejería de Salud y Familias de la Junta de Andalucía (projects noº PEMP0205-2020 FEDER funds), and by the Chair “Doctors Galera-Requena in cancer stem cell research” (CMC-CTS963). J.L.P. (Ref. FPU15/03682) acknowledge the MICIU for providing a PhD fellowship (FPU).info:eu-repo/semantics/publishedVersio

A review of mycotoxins in food and feed products in Portugal and estimation of probable daily intakes

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that occur naturally in agricultural commodities worldwide. Aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone, trichothecenes and ergot alkaloids are presently the most important for food and feed safety. These compounds are produced by several species that belong to the Aspergillus, Penicillium, Fusarium and Claviceps genera and can be carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic and immunosuppressant. Human and animal exposure to mycotoxins is generally assessed by taking into account data on the occurrence of mycotoxins in food and feed as well as data on the consumption patterns of the concerned population. This evaluation is crucial to support measures to reduce consumer exposure to mycotoxins. This work reviews the occurrence and levels of mycotoxins in Portuguese food and feed to provide a global overview of this issue in Portugal. With the information collected, the exposure of the Portuguese population to those mycotoxins is assessed, and the estimated dietary intakes are presented.Lu ıs Abrunhosa, H ector Morales, C elia Soares, and Thalita Calado received support through grants SFRH/BPD/43922/ 2008, SFRH/BPD/38011/2007, SFRH/BD/37264/2007 and SFRH/BD/79364/2011, respectively, from the Fundac¸ ao para ~ a Ciencia e Tecnologia ^ –FCT, Portugal