Contribution of extracellular vesicles for the pathogenesis of retinal diseases: shedding light on blood-retinal barrier dysfunction.

Retinal degenerative diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), loom as threats to vision, causing detrimental effects on the structure and function of the retina. Central to understanding these diseases, is the compromised state of the blood-retinal barrier (BRB), an effective barrier that regulates the influx of immune and inflammatory components. Whether BRB breakdown initiates retinal distress, or is a consequence of disease progression, remains enigmatic. Nevertheless, it is an indication of retinal dysfunction and potential vision loss.The intricate intercellular dialogues among retinal cell populations remain unintelligible in the complex retinal milieu, under conditions of inflammation and oxidative stress. The retina, a specialized neural tissue, sustains a ceaseless demand for oxygen and nutrients from two vascular networks. The BRB orchestrates the exchange of molecules and fluids within this specialized region, comprising the inner BRB (iBRB) and the outer BRB (oBRB). Extracellular vesicles (EVs) are small membranous structures, and act as messengers facilitating intercellular communication in this milieu.EVs, both from retinal and peripheral immune cells, increase complexity to BRB dysfunction in DR and AMD. Laden with bioactive cargoes, these EVs can modulate the retinal microenvironment, influencing disease progression. Our review delves into the multifaceted role of EVs in retinal degenerative diseases, elucidating the molecular crosstalk they orchestrate, and their microRNA (miRNA) content. By shedding light on these nanoscale messengers, from their biogenesis, release, to interaction and uptake by target cells, we aim to deepen the comprehension of BRB dysfunction and explore their therapeutic potential, therefore increasing our understanding of DR and AMD pathophysiology.

Molecular Pharmacology in Diabetes.

This Special Issue highlights the key molecules and molecular signaling pathways associated with diabetes and its multifaceted complications [...].

Evaluation of patients' satisfaction with domiciliary biological treatment in severe asthma: a Portuguese survey.

Dear Editor, According to GINA, severe asthma is defined as uncontrolled asthma, despite therapy adherence with an optimized high dose of inhaled corticosteroid plus long-acting ß2-agonist coupled with management of modifiable factors/comorbidities, that worsens when this treatment is decreased. It affects a significant portion of asthmatic patients and imposes a high risk of exacerbations and mortality, which are associated with significant healthcare costs and psychosocial impact...

New advances in endocrine hypertension: from genes to biomarkers.

Hypertension (HT) is a major cardiovascular risk factor that affects 10% to 40% of the general population in an age-dependent manner. Detection of secondary forms of HT is particularly important because it allows the targeted management of the underlying disease. Among hypertensive patients, the prevalence of endocrine HT reaches up to 10%. Adrenal diseases are the most frequent cause of endocrine HT and are associated with excess production of mineralocorticoids (mainly primary aldosteronism), glucocorticoids (Cushing syndrome), and catecholamines (pheochromocytoma). In addition, a few rare diseases directly affecting the action of mineralocorticoids and glucocorticoids in the kidney also lead to endocrine HT. Over the past years, genomic and genetic studies have allowed improving our knowledge on the molecular mechanisms of endocrine HT. Those discoveries have opened new opportunities to transfer knowledge to clinical practice for better diagnosis and specific treatment of affected subjects. In this review, we describe the physiology of adrenal hormone biosynthesis and action, the clinical and biochemical characteristics of different forms of endocrine HT, and their underlying genetic defects. We discuss the impact of these discoveries on diagnosis and management of patients, as well as new perspectives related to the use of new biomarkers for improved patient care.

Disturbed Matrix Metalloproteinases Activity in Age-Related Macular Degeneration.

Matrix metalloproteinases (MMPs) are a tightly regulated family of proteolytic enzymes that break down extracellular matrix (ECM) and basement membrane components. Because it is associated with development, morphogenesis, tissue remodeling, and repair, ECM remodeling is an important mechanism. MMPs are thought to act as a double-edged sword, as they contribute to maintaining photoreceptors/retinal pigment epithelium (RPE)/Bruch's membrane (BM)/choroid complex homeostasis and also contribute to the onset and progression of age-related macular degeneration (AMD). Polymorphisms and/or altered expression in MMPs and their tissue inhibitors (TIMPs) are associated with age-related macular degeneration (AMD). Here, we review the evidence for MMPs' role in the onset and progression of AMD via addressing their regulation and TIMPs' significant regulatory functions.

Immune Fingerprint in Diabetes: Ocular Surface and Retinal Inflammation.

Diabetes is a prevalent global health issue associated with significant morbidity and mortality. Diabetic retinopathy (DR) is a well-known inflammatory, neurovascular complication of diabetes and a leading cause of preventable blindness in developed countries among working-age adults. However, the ocular surface components of diabetic eyes are also at risk of damage due to uncontrolled diabetes, which is often overlooked. Inflammatory changes in the corneas of diabetic patients indicate that inflammation plays a significant role in diabetic complications, much like in DR. The eye's immune privilege restricts immune and inflammatory responses, and the cornea and retina have a complex network of innate immune cells that maintain immune homeostasis. Nevertheless, low-grade inflammation in diabetes contributes to immune dysregulation. This article aims to provide an overview and discussion of how diabetes affects the ocular immune system's main components, immune-competent cells, and inflammatory mediators. By understanding these effects, potential interventions and treatments may be developed to improve the ocular health of diabetic patients.

Improvement of Glycaemia and Endothelial Function by a New Low-Dose Curcuminoid in an Animal Model of Type 2 Diabetes.

Curcumin has been suggested as a promising treatment for metabolic diseases, but the high doses required limit its therapeutic use. In this study, a new curcuminoid is synthesised to increase curcumin anti-inflammatory and antioxidant potential and to achieve hypoglycaemic and protective vascular effects in type 2 diabetic rats in a lower dose. In vitro, the anti-inflammatory effect was determined through the Griess reaction, and the antioxidant activity through ABTS and TBARS assays. In vivo, Goto-Kakizaki rats were treated for 2 weeks with the equimolar dose of curcumin (40 mg/kg/day) or curcuminoid (52.4 mg/kg/day). Fasting glycaemia, insulin tolerance, plasma insulin, insulin signalling, serum FFA, endothelial function and several markers of oxidative stress were evaluated. Both compounds presented a significant anti-inflammatory effect. Moreover, the curcuminoid had a marked hypoglycaemic effect, accompanied by higher GLUT4 levels in adipose tissue. Both compounds increased NO-dependent vasorelaxation, but only the curcuminoid exacerbated the response to ascorbic acid, consistent with a higher decrease in vascular oxidative and nitrosative stress. SOD1 and GLO1 levels were increased in EAT and heart, respectively. Altogether, these data suggest that the curcuminoid developed here has more pronounced effects than curcumin in low doses, improving the oxidative stress, endothelial function and glycaemic profile in type 2 diabetes.

Unraveling the Photodynamic Activity of Cationic Benzoporphyrin-Based Photosensitizers against Bladder Cancer Cells.

In this study, we report the preparation of new mono-charged benzoporphyrin complexes by reaction of the appropriate neutral benzoporphyrin with (2,2'-bipyridine)dichloroplatinum(II) and of the analogs' derivatives synthesized through alkylation of the neutral scaffold with iodomethane. All derivatives were incorporated into polyvinylpyrrolidone (PVP) micelles. The ability of the resultant formulations to generate reactive oxygen species was evaluated, mainly the singlet oxygen formation. Then, the capability of the PVP formulations to act as photosensitizers against bladder cancer cells was assessed. Some of the studied formulations were the most active photosensitizers causing a decrease in HT-1376 cells' viability. This creates an avenue to further studies related to bladder cancer cells.

Highly Efficient Singlet Oxygen Generators Based on Ruthenium Phthalocyanines: Synthesis, Characterization and in vitro Evaluation for Photodynamic Therapy.

Invited for the cover of this issue is the group of Torres at the University of Madrid. The image of the cover of this issue depicts cancer cells being destroyed by reactive singlet oxygen produced by ruthenium phthalocyanine glycoconjugates under red light. The work, developed at the Universities of Madrid, Aveiro, Lisbon and Coimbra, describes ruthenium phthalocyanines as powerful bladder cancer PDT agents. Read the full text of the article at 10.1002/chem.201903546.

Highly Efficient Singlet Oxygen Generators Based on Ruthenium Phthalocyanines: Synthesis, Characterization and in vitro Evaluation for Photodynamic Therapy.

The synthesis of ruthenium(II) phthalocyanines (RuPcs) endowed with one carbohydrate unit-that is, glucose, galactose and mannose-and a dimethylsulfoxide (DMSO) ligand at the two axial coordination sites, respectively, is described. Two series of compounds, one unsubstituted at the periphery, and the other one bearing eight PEG chains at the isoindole meta-positions, have been prepared. The presence of the axial DMSO unit significantly increases the phthalocyanine singlet oxygen quantum yields, related to other comparable RuPcs. The compounds have been evaluated for PDT treatment in bladder cancer cells. In vitro studies have revealed high phototoxicity for RuPcs unsubstituted at their periphery. The phototoxicity of PEG-substituted RuPcs has been considerably improved by repeated light irradiation. The choice of the axial carbohydrate introduced little differences in the cellular uptake for both series of photosensitizers, but the phototoxic effects were considerably higher for compounds bearing mannose units.

Caveolin-1 Modulation Increases Efficacy of a Galacto-Conjugated Phthalocyanine in Bladder Cancer Cells Resistant to Photodynamic Therapy.

Photodynamic therapy (PDT) has demonstrated encouraging anticancer therapeutic results, but the current clinically approved photosensitizers (PSs) are not ideal in the treatment of bladder cancer. Conventional PSs have low selectivity to the bladder tumor tissue and induce toxicity or bystander effects on nontumor urothelium. Previous studies demonstrated that the use of galactose-photosensitizer (PS) conjugates is a more selective method of delivering PDT-mediated toxicity due to their ability to recognize carbohydrate-binding domains overexpressed in bladder tumors. Using patient-derived bladder tumor specimens cultured ex vivo and bladder cancer cell lines with different PDT sensitivity, we find that a galactose-phthalocyanine (PcGal16) accumulates in bladder tumors expressing galactose-binding proteins and internalizes through an endocytic process. The endocytosis mechanism is cell line-dependent. In HT-1376 bladder cancer lines resistant to PDT, depletion of caveolin-1-the main structural protein of caveolae structures-increased the amount of sugar-binding proteins, i.e. GLUT1, at the cell membrane resulting in an improved PcGal16 uptake and PDT efficacy. These data show the potential of ex vivo cultures of bladder cancer, that ideally could mimic the original microenvironment, in screening galacto-PDT agents. Additionally, our studies demonstrate that PDT efficacy in bladder cancer depends on the endocytic mechanisms that regulate PS accumulation and internalization in cancer cells.

Synthesis, Characterization and Photodynamic Activity against Bladder Cancer Cells of Novel Triazole-Porphyrin Derivatives.

Novel triazole-porphyrin derivatives (TZ-PORs) were synthesized through the Heck reaction and then incorporated into polyvinylpyrrolidone (PVP) micelles. After verifying that this incorporation did not compromise the photophysical and chemical features of TZ-PORs as photosensitizers, the phototoxicity of the formulations towards cancer cells was screened. Biological studies show high photodynamic activity of all PVP-TZ-POR formulations against a bladder cancer cell line with a particular highlight to PVP-TZ-POR 7e and 7f that are able to significantly reduce HT-1376 cell viability, while they had no effect on control ARPE-19 cells.

Photoimmunoconjugates: novel synthetic strategies to target and treat cancer by photodynamic therapy.

Photodynamic therapy (PDT) combines a photosensitizer (PS) with the physical energy of non-ionizing light to trigger cell death pathways. PDT has potential as a therapeutic modality to be used in alternative or in combination with other conventional cancer treatment protocols (e.g. surgery, chemotherapy and radiotherapy). Still, due to the lack of specificity of the current PSs to target the tumor cells, several studies have exploited their conjugation with targeting moieties. PSs conjugated with antibodies (Abs) or their fragments, able to bind antigens overexpressed in the tumors, have demonstrated potential in PDT of tumors. This review provides an overview of the most recent advances on photoimmunoconjugates (PICs) for cancer PDT, which involve the first and second-generation PSs conjugated to Abs. This is an update of our previous review "Antibodies armed with photosensitizers: from chemical synthesis to photobiological applications", published in 2015 in Org. Biomol. Chem.

Multicharged Phthalocyanines as Selective Ligands for G-Quadruplex DNA Structures.

The stabilization of G-Quadruplex DNA structures by ligands is a promising strategy for telomerase inhibition in cancer therapy since this enzyme is responsible for the unlimited proliferation of cancer cells. To assess the potential of a compound as a telomerase inhibitor, selectivity for quadruplex over duplex DNA is a fundamental attribute, as the drug must be able to recognize quadruplex DNA in the presence of a large amount of duplex DNA, in the cellular nucleus. By using different spectroscopic techniques, such as ultraviolet-visible, fluorescence and circular dichroism, this work evaluates the potential of a series of multicharged phthalocyanines, bearing four or eight positive charges, as G-Quadruplex stabilizing ligands. This work led us to conclude that the existence of a balance between the number and position of the positive charges in the phthalocyanine structure is a fundamental attribute for its selectivity for G-Quadruplex structures over duplex DNA structures. Two of the studied phthalocyanines, one with four peripheral positive charges (ZnPc1) and the other with less exposed eight positive charges (ZnPc4) showed high selectivity and affinity for G-Quadruplex over duplex DNA structures and were able to accumulate in the nucleus of UM-UC-3 bladder cancer cells.

Carbon-1 versus Carbon-3 Linkage of d-Galactose to Porphyrins: Synthesis, Uptake, and Photodynamic Efficiency.

The use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as porphyrins provides a promising strategy to improve the selectivity and cell uptake of the photosensitizers, enhancing the overall photosensitizing efficacy. Most porphyrin-carbohydrate conjugates are linked via the carbon-1 position of the carbohydrate because this is the most synthetically accessible approach. Previous studies suggest that carbon-1 galactose derivatives show diminished binding since the hydroxyl group in the carbon-1 position of the sugar is a hydrogen bond acceptor in the galectin-1 sugar binding site. We therefore synthesized two isomeric porphyrin-galactose conjugates using click chemistry: one linked via the carbon-1 of the galactose and one linked via carbon-3. Free base and zinc analogs of both conjugates were synthesized. We assessed the uptake and photodynamic therapeutic (PDT) activity of the two conjugates in both monolayer and spheroidal cell cultures of four different cell lines. For both the monolayer and spheroid models, we observe that the uptake of both conjugates is proportional to the protein levels of galectin-1 and the uptake is suppressed after preincubation with an excess of thiogalactose, as measured by fluorescence spectroscopy. Compared to that of the carbon-1 conjugate, the uptake of the carbon-3 conjugate was greater in cell lines containing high expression levels of galectin-1. After photodynamic activation, MTT and lactate dehydrogenase assays demonstrated that the conjugates induce phototoxicity in both monolayers and spheroids of cancer cells.

Allergic contact dermatitis caused by dexpanthenol-Probably a frequent allergen.

BACKGROUND: Panthenol (synonym: dexpanthenol), the alcohol analogue of panthothenic acid, is frequently included in moisturizers, wound-healing agents, and other cosmetics, and has been shown to be responsible for allergic contact dermatitis (ACD). OBJECTIVES: To evaluate the frequency of ACD caused by dexpanthenol, and to characterize reactive patients. METHODS: We retrospectively reviewed the files of patients patch tested between 2009 and 2017 in the Department of Dermatology of the Coimbra's University Hospital and describe patients who reacted to dexpanthenol 5% pet., tested initially in a cosmetic/vehicle series and in the last 3 years in consecutive patients. RESULTS: Among 2171 patients, 26 (1.2%) had positive patch test reactions to dexpanthenol, mostly patients tested for chronic eczema (88.5%, n = 23), either widespread (5), or localized to the hands (5), face (4), or legs (7). Relevance could be traced in 20 patients (76.9%), related to the use of Bepanthene cream (15), moisturizers (3), topical medications (1), and a shampoo (1). Twenty-five of 26 patients (96.2%) reacted to several other allergens, mostly ingredients of cosmetic or pharmaceutical products. CONCLUSIONS: Although ACD caused by dexpanthenol is considered to be rare, it may be frequently overlooked. As we found a relatively high frequency of relevant cases, in agreement with a previous study, the inclusion of dexpanthenol in patch test series, at least in cosmetic and topical drug series, is encouraged.

Mitochondria-Targeted Photodynamic Therapy with a Galactodendritic Chlorin to Enhance Cell Death in Resistant Bladder Cancer Cells.

Here, we report the rational design of a new third-generation photosensitizer (PS), a chlorin conjugated with galactodendritic units, ChlGal8, to improve the effectiveness of bladder cancer treatment. ChlGal8 shows better photochemical and photophysical properties than a recently reported homologous porphyrin, PorGal8. In addition to inheriting excellent photostability, the ability to generate singlet oxygen, and the ability to interact with the proteins galectin-1 and human serum albumin (HSA), ChlGal8 exhibits high absorption in the red region of the electromagnetic spectrum. In vitro studies of anticancer activity of ChlGal8 revealed that once this PS is taken up by UM-UC-3 bladder cancer cells, it induces high cytotoxicity after a single dose of light irradiation. In HT-1376 bladder cancer cells resistant to therapy, a second light irradiation treatment enhanced in vitro and in vivo photodynamic efficacy. The enhanced phototoxicity in HT-1376 cancer cells seems to be due to the ability of ChlGal8 to accumulate in the mitochondria, via facilitative glucose transporter 1 (GLUT1), in the period between single and repeated irradiation. A photodynamic therapy (PDT) regimen using an extra dose of light irradiation and ChlGal8 as PS represents a promising strategy in treating resistant cancers in a clinical setting.

Dipeptidyl peptidase-IV inhibition prevents blood-retinal barrier breakdown, inflammation and neuronal cell death in the retina of type 1 diabetic rats.

Diabetic retinopathy, a leading cause of vision loss in working-age population, is often associated with inflammation and apoptosis. We have previously reported that sitagliptin, a DPP-IV inhibitor, exerts beneficial effects in the retina of type 2 diabetic animals. The present study aimed to evaluate whether sitagliptin can exert protective effects in the retina of type 1 diabetic animals by a mechanism independent of insulin secretion and glycemia normalization. Streptozotocin-induced diabetic rats were treated orally with sitagliptin (5mg/kg/day) for the last two weeks of 4 weeks of diabetes. Sitagliptin treatment did not change the weight and glucose, HbA1c or insulin levels. However, it prevented the diabetes-induced increase in DPP-IV/CD26 activity and levels in serum and retina. Sitagliptin also prevented the increase in blood-retinal barrier (BRB) permeability and inhibited the changes in immunoreactivity and endothelial subcellular distribution of occludin, claudin-5 and ZO-1 proteins induced by diabetes. Furthermore, sitagliptin decreased the retinal inflammatory state and neuronal apoptosis. Sitagliptin inhibited the BRB breakdown in a type 1 diabetic animal model, by a mechanism independent of normalization of glycemia, by preventing changes in tight junctions (TJs) organization. Sitagliptin also exerted protective effects against inflammation and pro-apoptotic state in the retina of diabetic rats. Altogether, these results suggest that sitagliptin might be envisaged to be used to prevent or delay some of the alterations associated with the development of diabetic retinopathy.

Sonic Hedgehog mutations are not a common cause of congenital hypopituitarism in the absence of complex midline cerebral defects.

CONTEXT AND OBJECTIVE: Sonic Hedgehog (SHH) and GLI2, an obligatory mediator of SHH signal transduction, are holoprosencephaly (HPE)-associated genes essential in pituitary formation. GLI2 variants have been found in patients with congenital hypopituitarism without complex midline cerebral defects (MCD). However, data on the occurrence of SHH mutations in these patients are limited. We screened for SHH and GLI2 mutations or copy number variations (CNV) in patients with congenital hypopituitarism without MCD or with variable degrees of MCD. PATIENTS AND METHODS: Detailed data on clinical, laboratory and neuroimaging findings of 115 patients presenting with congenital hypopituitarism without MCD, septo-optic dysplasia or HPE were analysed. The SHH and GLI2 genes were directly sequenced, and the presence of gene CNV was analysed by multiplex ligation-dependent probe amplification (MLPA). RESULTS: Anterior pituitary deficiency was found in 74% and 53% of patients with SOD or HPE, respectively. Diabetes insipidus was common in patients with HPE (47%) but infrequent in patients with congenital hypopituitarism or SOD (7% and 8%, respectively). A single heterozygous nonsense SHH mutation (p.Tyr175Ter) was found in a patient presenting with hypopituitarism and alobar HPE. No other SHH mutations or CNV were found. Nine GLI2 variations (8 missense and 1 frameshift) including a homozygous and a compound heterozygous variation were found in patients with congenital hypopituitarism or SOD, but not in HPE patients. No GLI2 CNV were found. CONCLUSION: SHH mutations or copy number variations are not a common cause of congenital hypopituitarism in patients without complex midline cerebral defects. GLI2 variants are found in some patients with congenital hypopituitarism without complex midline cerebral defects or septo-optic dysplasia. However, functional analyses of these variants are needed to strengthen genotype-phenotype relationship.

Bilateral Idiopathic Adrenal Hyperplasia: Genetics and Beyond.

Bilateral adrenal hyperplasia currently accounts for up to 2 thirds of cases of primary aldosteronism. As such, it represents a major opportunity for targeted medical management as opposed to unilateral surgically correctable forms of the disease. Although the majority of cases of primary aldosteronism are sporadic, bilateral adrenal hyperplasia may occur in the context of familial hyperaldosteronism where it is associated with specific germline mutations. Over the past 5 years, impressive progress has been made in our understanding of the genetic basis underlying primary aldosteronism, allowing us to identify and characterize new familial forms of the disease and to understand the mechanisms involved in the formation of aldosterone producing adenoma. In contrast, our knowledge of the genetic contribution to the development of bilateral adrenal hyperplasia, and in a larger context, to renin and aldosterone levels in the general population, is still poor. This review summarizes our current knowledge on the genetics of bilateral adrenal hyperplasia and addresses some open questions to be addressed by future research. In particular, genome-wide association studies in large populations may provide clues to understanding the genetic susceptibility underlying the development of primary aldosteronism.