Search

Purpose: Meibomian gland dysfunction (MGD) is a major cause of signs and symptoms related to dry eyes (DE) and eyelid inflammation. We investigated the composition of human tears by metabolomic approaches in patients with aqueous tear deficiency and MGD. Methods: Participants in this prospective, case-control pilot study were split into patients with aqueous tear deficiency and MGD (DE-MGD [n = 15]) and healthy controls (CG; n = 20). Personal interviews, ocular surface disease index (OSDI), and ophthalmic examinations were performed. Reflex tears collected by capillarity were processed to 1H nuclear magnetic resonance (NMR) spectroscopy and quantitative data analysis to identify molecules by spectra comparison to library entries of purified standards and/or unknown entities. Statistical analyses were made by the SPSS 22.0 program. Results: Chemometric analysis and 1H NMR spectra comparison revealed the presence of 60 metabolites in tears. Differentiating features were evident in the NMR spectra of the 2 clinical groups, characterized by significant upregulation of phenylalanine, glycerol, and isoleucine, and downregulation of glycoproteins, leucine, and –CH3 lipids, as compared to the CG. The 1H NMR metabolomic analyses of human tears confirmed the applicability of this platform with high predictive accuracy/reliability. Conclusions: Our key distinctive findings support that DE-MGD induces tear metabolomics profile changes. Metabolites contributing to a higher separation from the CG can presumably be used, in the foreseeable future, as DE-MGD biomarkers for better managing the diagnosis and therapy of this disease.

Resumen Objetivo Analizar la seguridad y efectividad de la administracion oral de un suplemento comercializado que contiene acido alfa-R-lipoico, taurina, vitaminas C y E, luteina, zeaxantina, zinc, cobre y acido docosahexaenoico en pacientes con glaucoma primario de angulo abierto (GPAA) y sujetos control. Material y metodos Se realizo un estudio prospectivo de casos y controles que incluyo 30 participantes de ambos sexos, divididos en: pacientes con GPAA (n = 15) y sujetos sanos (GC; n = 15) asignados a tomar durante 6 meses los preparados NuaDHA Vision® (1 comp/dia) + NuaDHA 1000 (2 comps/dia). Los participantes fueron entrevistados, examinados oftalmologicamente, extrayendo sangre periferica que fue procesada para analitica convencional y determinacion de actividades prooxidante (malonildialdehido) y estado antioxidante total. El analisis estadistico se realizo mediante el programa SPSS 22.0. Resultados Tras 6 meses de suplementacion, los niveles plasmaticos de estado antioxidante total aumentaron significativamente (1,073 ± 0,090 mM vs. 1,276 ± 0,107 mM; p = 0,028), disminuyendo en paralelo los de malonildialdehido (7,066 ± 1,070 μM vs. 2,761 ± 0,462 μM; p = 0,005) en el GGPAA. El malonildialdehido tambien descendio significativamente en el GC (6,17 ± 1,336 vs. 2,51 ± 0,391; p = 0,028). Los pacientes con GPAA mostraron valores del test de Schirmer notablemente mayores (20-30%) y mejoraron subjetivamente los signos/sintomas de ojo seco, frente a los resultados del GC. Conclusiones Las formulaciones que contienen vitaminas antioxidantes, acido alfa-R-lipoico y acido docosahexaenoico administradas durante 6 meses consecutivos contrarrestaron el estres oxidativo, y estabilizaron los parametros morfologicos/funcionales de la superficie ocular y del glaucoma, sin presentar efectos adversos o intolerancias.

Objective To analyse the safety and effectiveness of the oral administration of a commercialised supplement containing R-alpha lipoic acid (ALA), taurine, vitamins C and E, lutein, zeaxanthin, zinc, copper and docosahexaenoic acid (DHA), in patients with primary open angle glaucoma (POAG), and in control subjects. Material and methods A prospective study of cases and controls was carried out, including 30 participants of both genders that were divided into: POAG Group (n = 15) and a control group (CG; n = 15), assigned to the oral intake of NuaDHA preparations Vision® (1 pill/day) + NuaDHA 1000 (2 pills/day) for 6 months. Participants were interviewed, ophthalmologically examined, and peripheral blood was taken for routine analysis and the determination of the pro-oxidant [malondialdehyde (MDA)] and total antioxidant status (TAS). Statistical analysis was performed using the SPSS 22.0 program. Results After 6 months of supplementation, there was a significant increase in the plasma TAS (1.073 ± 0.090 mM vs 1.276 ± 0.107 mM, P = .028), along with a parallel decrease in MDA (7.066 ± 1.070 μM vs 2.771 ± 0.462 μM, P = .005) in the POAG group. The MDA also decreased in the control group (6.17 ± 1.336 vs. 2.51 ± 0.391, P = .028). The Schirmer test improved (20–30%) and the subjective dry eye signs/symptoms noticeably decreased in the POAG group versus the CG. Conclusions Formulations containing antioxidant vitamins, ALA and DHA, administered for 6 consecutive months, counteracted the oxidative stress by further stabilising the morphological/functional parameters of both the ocular surface and the glaucoma, without presenting with adverse effects or intolerances.

Purpose: To assess the changes in retinal morphology in a rat model of chronic glaucoma induced by ocular hypertension. Methods: Intraocular pressure (IOP) was surgically increased through weekly injections of sodium hyaluronate (HYA) in the anterior eye chamber of the left eye of male Wistar rats, whereas the right eyes were sham operated (salt solution). During the 10-week experimental period, IOP was measured weekly with a rebound tonometer. Retinal cryosections were prepared for histological/immunohistochemical analysis and morphometry. Results: IOP was higher in HYA-treated eyes than in sham-operated eyes along the 10-week period, which was significant from the fourth to the nineth week. Ocular hypertension in HYA-treated eyes was associated with morphologic and morphometric changes in bipolar cells, ON-OFF direction-selective ganglion cells, ON/OFF starburst amacrine cells, and inner plexiform layer sublamina. Conclusions: Serial HYA treatment in the rat anterior eye chamber results in mild-to-moderate elevated and sustained IOP and ganglion cell death, which mimics most human open-angle glaucoma hallmarks. The reduced number of direction-selective ganglion cells and starburst amacrine cells accompanied by a deteriorated ON/OFF plexus in this glaucoma model could lend insight to the abnormalities in motion perception observed in patients with glaucoma. Supported by the Spanish Ministry of Science and Innovation (MICINN-FEDER PID2019-106230RB-I00), 2), Institute of Health Carlos III (General Subdirection of Networks and Cooperative Research Centers RETICs OFTARED 2012-2021) co-financed by the European Regional Development fund (RD16/0008/0016 [Alicante] and RD16/0008/0022 [Valencia]), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), Generalitat Valenciana (IDIFEDER/2017/064), and the Research funds from both Department of Surgery (Ophthalmology Unit) and Cellular-Molecular Ophthalmobiology Group of the University of Valencia (Spain).

Este artículo se encuentra disponible en la siguiente URL: https://www.mdpi.com/2077-0383/10/11/2227 En esta investigación también participan: Mar Valero Vello, Silvia M. Sanz González, José E. O'Connor, David Galarreta Mira, María D. Pinazo-Durán y Vicente Zanón Moreno. Este artículo pertenece al número especial "Recent Clinical Research on Glaucoma". Glaucoma has no cure and is a sight-threatening neurodegenerative disease affecting more than 100 million people worldwide, with primary open angle glaucoma (POAG) being the most globally prevalent glaucoma clinical type. Regulation of gene expression and gene networks, and its multifactorial pathways involved in glaucoma disease are landmarks for ophthalmic research. MicroRNAs (miRNAs/miRs) are small endogenous non-coding, single-stranded RNA molecules (18–22 nucleotides) that regulate gene expression. An analytical, observational, case-control study was performed in 42 patients of both sexes, aged 50 to 80 years, which were classified according to: (1) suffering from ocular hypertension (OHT) but no glaucomatous neurodegeneration (ND) such as the OHT group, or (2) have been diagnosed of POAG such as the POAG group. Participants were interviewed for obtaining sociodemographic and personal/familial records, clinically examined, and their tear samples were collected and frozen at 80 C until processing for molecular-genetic assays. Tear RNA extraction, libraries construction, and next generation sequencing were performed. Here, we demonstrated, for the first time, the differential expression profiling of eight miRNAs when comparing tears from the OHT versus the POAG groups: the miR-26b-5p, miR-152-3p, miR-30e-5p, miR-125b-2-5p, miR-224-5p, miR-151a-3p, miR-1307-3p, and the miR-27a-3p. Gene information was set up from the DIANA-TarBase v7, DIANA-microT-CDS, and TargetScan v7.1 databases. To build a network of metabolic pathways, only genes appearing in at least four of the following databases: DisGeNet, GeneDistiller, MalaCards, OMIM PCAN, UniProt, and GO were considered. We propose miRNAs and their target genes/signaling pathways as candidates for a better understanding of the molecular-genetic bases of glaucoma and, in this way, to gain knowledge to achieve optimal diagnosis strategies for properly identifying HTO at higher risk of glaucoma ND. Further research is needed to validate these miRNAs to discern the potential role as biomarkers involved in oxidative stress, immune response, and apoptosis for the diagnosis and/or prognosis of OHT and the prevention of glaucoma ND.

Adherence to a healthy diet offers a valuable intervention to compete against the increasing cases of ocular diseases worldwide, such as dry eye disorders, myopia progression, cataracts, glaucoma, diabetic retinopathy, or age macular degeneration. Certain amounts of micronutrients must be daily provided for proper functioning of the visual system, such as vitamins, carotenoids, trace metals and omega-3 fatty acids. Among natural foods, the following have to be considered for boosting eye/vision health: fish, meat, eggs, nuts, legumes, citrus fruits, nuts, leafy green vegetables, orange-colored fruits/vegetables, olives-olive oil, and dairy products. Nutritional supplements have received much attention as potential tools for managing chronic-degenerative ocular diseases. A systematic search of PubMed, Web of Science, hand-searched publications and historical archives were performed by the professionals involved in this study, to include peer-reviewed articles in which natural food, nutrient content, and its potential relationship with ocular health. Five ophthalmologists and two researchers collected the characteristics, quality and suitability of the above studies. Finally, 177 publications from 1983 to 2021 were enclosed, mainly related to natural food, Mediterranean diet (MedDiet) and nutraceutic supplementation. For the first time, original studies with broccoli and tigernut (chufa de Valencia) regarding the ocular surface dysfunction, macular degeneration, diabetic retinopathy and glaucoma were enclosed. These can add value to the diet, counteract nutritional defects, and help in the early stages, as well as in the course of ophthalmic pathologies. The main purpose of this review, enclosed in the Special Issue “Health Benefits and Nutritional Quality of Fruits, Nuts and Vegetables,” is to identify directions for further research on the role of diet and nutrition in the eyes and vision, and the potential antioxidant, anti-inflammatory and neuroprotective effects of natural food (broccoli, saffron, tigernuts and walnuts), the Mediterranean Diet, and nutraceutic supplements that may supply a promising and highly affordable scenario for patients at risk of vision loss. This review work was designed and carried out by a multidisciplinary group involved in ophthalmology and ophthalmic research and especially in nutritional ophthalmology.

Primary open-angle glaucoma (POAG) is a paramount cause of irreversible visual disability worldwide. We focus on identifying clinical and molecular facts that may help elucidating the pathogenic mechanisms of the disease. By using ophthalmological approaches (biomicroscopy, ocular fundus, optical coherence tomography, and perimetry) and experimental tests (enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), and Western blot/immunoblotting) directed to evaluate the oxidative stress, inflammation, apoptosis, and neurodegeneration processes, we gather information to build a network of data to perform a computational bioinformatics analysis. Our results showed strong interaction of the above players and its downstream effectors in POAG pathogenesis. In conclusion, specific risk factors were identified, and molecules involved in multiple pathways were found in relation to anterior and posterior eye segment glaucoma changes, pointing to new theranostic challenges for better managing POAG progression.

Resumen Objetivo Identificar genes implicados en los mecanismos patogenicos de la retinopatia diabetica no proliferante, como estres oxidativo, alteracion de la matriz extracelular y/o apoptosis, para valorar el riesgo de desarrollo de la misma en una poblacion de diabeticos tipo 2 (DM2). Material y metodos Estudio de casos y controles en 81 participantes del Estudio Valencia sobre Retinopatia Diabetica (EVRD), de ambos sexos y con edades comprendidas entre los 25 y 85 anos, clasificados en: 1) grupo DM2 (n = 49), con RD (+RD; n = 14) y sin RD (−RD; n = 35), y 2) grupo control (GC; n = 32). Se realizo entrevista personal, examen oftalmologico estandarizado y extraccion de sangre que se proceso para analizar el ADN y determinar la expresion de: TP53, MMP9 y SLC23A2 en todos los participantes. El programa estadistico utilizado fue el SPSS v22.0. Resultados Los genes TP53 y MMP9 aumentaron su expresion en el grupo DM2 respecto al GC, aunque solo de manera significativa el gen MMP9 (TP53: 10,40 ± 1,20 vs. 8,23 ± 1,36, p = 0,084; MMP9: 1,45 ± 0,16 vs. 0,95 ± 0,16, p = 0,036) y el gen SLC23A2 disminuyo significativamente sus niveles en DM2 vs. GC (5,58 ± 0,64 vs. 11,66 ± 1,90, p = 0,026). Al subdividir el grupo DM2 segun presencia de retinopatia, la expresion de los genes TP53, MMP9 y SLC23A2 mostro diferencias significativas entre los grupos DM2−RD, DM2+RD y GC (TP53: 9,95 ± 1,47 vs. 11,52 ± 2,05 vs. 8,23 ± 1,36, p = 0,038; MMP9: 1,47 ± 0,20 vs. 1,41 ± 0,27 vs. 0,95 ± 0,16, p = 0,021; SLC23A2: 5,61 ± 0,77 vs. 5,51 ± 1,21 vs. 11,66 ± 1,90, p = 0,018). Conclusiones Los genes reguladores de apoptosis (TP53) e integridad de la matriz extracelular (MMP9) podrian estar implicados en la susceptibilidad para el desarrollo/progresion de la RD, asi como el gen SLC232A2 (transportador del acido ascorbico) puede comportarse como protector del riesgo de padecer/progresar en la retinopatia.

Objective To identify genes involved in the pathogenic mechanisms of non-proliferative diabetic retinopathy (NPDR), among which include oxidative stress, extracellular matrix changes, and/or apoptosis, in order to evaluate the risk of developing this retinal disease in a type 2 diabetic (DM2) population. Material and methods A case–control study was carried out on 81 participants from the Valencia Study on Diabetic Retinopathy (VSDR) of both genders, with ages 25–85 years. They were classified into: (i) DM2 group (n = 49), with DR (+DR; n = 14) and without DR (−DR; n = 35), and (ii) control group (GC; n = 32). The protocols included a personal interview, standardized ophthalmological examination, and blood collection (to analyze the DNA for determining the gene expression (TP53, MMP9, and SLC23A2) in the study groups). Statistical analyses were performed using the SPSS v22.0 program. Results The TP53 and MMP9 genes showed a higher expression in the DM2 group compared to the GC, although the difference was only significant for the MMP9 gene (TP53: 10.40 ± 1.20 vs. 8.23 ± 1.36, p = 0.084; MMP9: 1.45 ± 0.16 vs. 0.95 ± 0.16, p = 0.036), and the SLC23A2 gene showed a significant lower expression in the DM2 vs. CG (5.58 ± 0.64 vs. 11.66 ± 1.90, p = 0.026). When sub-dividing the DM2 group according to the presence of retinopathy, the expression of the TP53, MMP9 and SLC23A2 genes showed significant differences between the DM2−RD, DM2+RD and GC groups (TP53: 9.95 ± 1.47 vs. 11.52 ± 2.05 vs. 8.23 ± 1.36, p = 0.038; MMP9: 1.47 ± 0.20 vs. 1.41 ± 0.27 vs. 0.95 ± 0.16, p = 0.021; SLC23A2: 5.61 ± 0.77 vs. 5.51 ± 1.21 vs. 11.66 ± 1.90, p = 0.018). Conclusions Genes involved in extracellular matrix integrity (MMP9) and/or apoptosis (TP53), could be considered potential markers of susceptibility to the development/progression of NPDR. Interestingly, the SLC232A2 gene (ascorbic acid transporter) can be considered a protector of the risk of the development/progression of the retinopathy.

The chapter is a review enclosed in the volume "Glaucoma: A pancitopatia of the retina and beyond." No cure exists for glaucoma. Knowledge on the molecular and cellular alterations underlying glaucoma neurodegeneration (GL-ND) includes innovative and path-breaking research on neuroinflammation and neuroprotection. A series of events involving immune response (IR), oxidative stress and gene expression are occurring during the glaucoma course. Uveitic glaucoma (UG) is a prevalent acute/chronic complication, in the setting of chronic anterior chamber inflammation. Managing the disease requires a team approach to guarantee better results for eyes and vision. Advances in biomedicine/biotechnology are driving a tremendous revolution in ophthalmology and ophthalmic research. New diagnostic and imaging modalities, constantly refined, enable outstanding criteria for delimiting glaucomatous neurodegeneration. Moreover, biotherapies that may modulate or inhibit the IR must be considered among the first-line for glaucoma neuroprotection. This review offers the readers useful and practical information on the latest updates in this regard.

Reactive oxygen species (ROS) overproduction and ROS-signaling pathways activation attack the eyes. We evaluated the oxidative stress (OS) and the effects of a daily, core nutritional supplement regimen containing antioxidants and omega 3 fatty acids (A/ω3) in type 2 diabetics (T2DM). A case-control study was carried out in 480 participants [287 T2DM patients with (+)/without (−) diabetic retinopathy (DR) and 193 healthy controls (CG)], randomly assigned to a daily pill of A/ω3. Periodic evaluation through 38 months allowed to outline patient characteristics, DR features, and classic/OS blood parameters. Statistics were performed by the SPSS 24.0 program. Diabetics displayed significantly higher circulating pro-oxidants (p = 0.001) and lower antioxidants (p = 0.0001) than the controls. Significantly higher plasma malondialdehyde/thiobarbituric acid reactive substances (MDA/TBARS, p = 0.006) and lower plasma total antioxidant capacity (TAC, p = 0.042) and vitamin C (0.020) was found in T2DM + DR versus T2DM-DR. The differential expression profile of solute carrier family 23 member 2 (SLC23A2) gene was seen in diabetics versus the CG (p = 0.001), and in T2DM + DR versus T2DM − DR (p &lt, 0.05). The A/ω3 regime significantly reduced the pro-oxidants (p &lt, 0.05) and augmented the antioxidants (p &lt, 0.05). This follow-up study supports that a regular A/ω3 supplementation reduces the oxidative load and may serve as a dietary prophylaxis/adjunctive intervention for patients at risk of diabetic blindness.

Purpose: To evaluate skin biocompatibility of a nighttime hydrating eyelid gel and possible ocular surface effects in contact lens users (CLU) and non-contact lens users (NCLU). The formulation is registered as a medical device as Tridocosahexaenoine-AOX® (TDHA-AOX) (a concentrated DHA triglyceride), containing also hyaluronic acid (HA). Methods: A prospective, randomized, masked clinical trial was performed with 62 participants of both sexes, aged 20–70 years, split into: (1) CLU (n = 30) and (2) NCLU (n = 32). All participants were instructed to apply a single dose of the moisturizing gel (containing TDHA-AOX and HA) nightly to the upper and inner eyelids of their right eye (RE) only, and during 2 consecutive weeks. Personal interviews, questionnaires, ophthalmic examinations and reflex tear collection were performed. Ophthalmological parameters included ocular surface response and contact lens status. Levels of satisfaction/adverse events were also recorded. Biochemical parameters included basal and final determination of pro-inflammatory mediator molecules in tear samples by multiplex analyses. Statistics were done by the SPSS 24.0 program. Results: The CLU group had higher OS dysfunction than NCLU, but overall clinical parameters (corneal staining, and Schirmer/FBUT tests) and OSDI scores showed significant improvement in CLU individuals as compared to the NCLU participants, at the end of study. CLDEQ-8 scores pinpointed significant amelioration in initial risk of developing DEs by applying eyelid gel. Multiplex analyses demonstrated significantly lower VEGF expression levels (p

Background: The aim of this study was to assess the expression of cytokines/chemokines in tears from patients with non-advanced primary open-angle glaucoma and patients with non-severe dry eye disease versus healthy controls. Methods: This prospective, observational cohort study enrolled patients with confirmed or suspected non-advanced primary open-angle glaucoma who received any prostaglandin analogue monotherapy for longer than 6 months, patients with non-severe dry eye disease, and healthy controls. Expression of interleukin-1β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, and interleukin-12; tumor necrosis factor α; vascular endothelial growth factor; granulocyte-macrophage colony-stimulating factor; and interferon γ was assessed. Results: 107 participants were enrolled (primary open-angle glaucoma, n = 41; dry eye disease, n = 30; and healthy controls, n = 36). Compared with healthy controls, interleukin-6 was significantly higher ( p = 0.0001) in patients with primary open-angle glaucoma and interleukin-1β ( p = 0.0144), interleukin-6 ( p Conclusion: Different cytokine/chemokine expression profiles in tears of patients with primary open-angle glaucoma and dry eye disease strongly suggest the involvement of a variety of signaling pathways in the pathogenesis of these ophthalmic processes.

We have studied the global risk of retinopathy in a Mediterranean population of type 2 diabetes mellitus (T2DM) patients, according to clinical, biochemical, and lifestyle biomarkers. The effects of the oral supplementation containing antioxidants/omega 3 fatty acids (A/ω3) were also evaluated. Suitable participants were distributed into two main groups: (1) T2DMG (with retinopathy (+DR) or without retinopathy (−DR)) and (2) controls (CG). Participants were randomly assigned (+A/ω3) or not (−A/ω3) to the oral supplementation with a daily pill of Nutrof Omega (R) for 18 months. Data collected including demographics, anthropometrics, characteristics/lifestyle, ophthalmic examination (best corrected visual acuity, ocular fundus photographs, and retinal thickness as assessed by optical coherence tomography), and blood parameters (glucose, glycosylated hemoglobin, triglycerides, malondialdehyde, and total antioxidant capacity) were registered, integrated, and statistically processed by the SPSS 15.0 program. Finally, 208 participants (130 diabetics (68 +DR/62 −DR) and 78 controls) completed the follow-up. Blood analyses confirmed that the T2DMG+DR patients had significantly higher oxidative stress(p<0.05), inflammatory(p<0.05), and vascular(p<0.001)risk markers than the T2DMG−DR and the CG. Furthermore, the A/ω3 oral supplementation positively changed the baseline parameters, presumptively by inducing metabolic activation and ameliorating the ocular health after 18 months of supplementation.

1. Tight control of cellular growth is essential to ensure normal tissue patterning and prevent pathological responses. Excessive vascular smooth muscle cell (VSMC) proliferation is associated with the pathophysiology of atherosclerosis and restenosis post-angioplasty. Thus, drug targeting of pathological VSMC growth may be a suitable therapeutic intervention in vascular proliferative diseases. 2. In the present study, we investigated the mechanisms underlying VSMC growth arrest induced by the pharmacological agent PCA-4230. Addition of PCA-4230 to cultured VSMCs blocked the induction of cyclin D1 and cyclin A expression normally seen in serum-restimulated cells. Moreover, PCA-4230 inhibited cyclin-dependent kinase 2 (CDK2) activity and abrogated hyperphosphorylation of the retinoblastoma (Rb) gene product. Similarly, PCA-4230-dependent growth arrest of transformed cell lines correlated with reduced level of cyclin D1 protein and inhibition of CDK2 activity. Consistent with these findings, PCA-4230 repressed serum-inducible cyclin A promoter activity, and overexpression of either cyclin D1 or E2F1 efficiently circumvented this inhibitory effect. Importantly, adenovirus-mediated overexpression of E2F1 restored S-phase entry in PCA-4230-treated VSMCs, demonstrating that PCA-4230 represses cyclin A gene expression and VSMC growth via inhibition of the cyclin D1/E2F pathway. 3. Because of its ability to inhibit the growth of human VSMCs and transformed cell lines, future studies are warranted to assess whether PCA-4230 may be a suitable therapeutic intervention for the treatment of hyperproliferative disorders, including cardiovascular disease and cancer.

Abnormal cellular proliferation is associated with the pathology of several diseases, including cancer, atherosclerosis and restenosis post-angioplasty. Therefore, anti-proliferative therapies may be a suitable approach to treat these disorders. Candidate targets for such strategies include specific components of the cell cycle machinery. Progression through the cell cycle in mammalian cells requires the activation of several cyclin-dependent protein kinases (CDKs) through their association with regulatory subunits called cyclins. Active CDK/cyclin holoenzymes phosphorylate cellular proteins including the retinoblastoma susceptibility gene product (pRb) and the related pocket proteins p107 and p130. Several compounds have been described that directly or indirectly inhibit the activity of CDKs, which results in a suppression of cell growth. In this review, we will discuss the use of drugs targeting CDKs and their therapeutic application in animal models and clinical trials.

Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study provides experimental evidence for the induction – shortly after isolation through the classical procedure – of strong oxidative stress that involves both oxygen-derived and NO-derived species. NO formation at this stage is due to the early activation of liver constitutive NO synthase (cNOS). Immunodetection of nitrated proteins provides direct evidence of endogenous peroxynitrite (PN) formation upon hepatocyte isolation. On the basis of the combined use of dihydrorhodamine 123 and NOS inhibitors, the analysis of the amount, time course and nature of the species involved supports the view that PN generated from cNOS-derived NO, while not affecting cell viability and hepatocyte monolayer development, is the main species likely responsible for the early biochemical injury commonly observed in hepatocyte cultures.

Insulin receptor substrate-2 (Irs2) mediates peripheral insulin action and is essential for retinal health. Previous investigations have reported severe photoreceptor degeneration and abnormal visual function in Irs2-deficient mice. However, molecular changes in the Irs2 − / − mouse retina have not been described. In this study, we examined retinal degenerative changes in neuronal and glial cells of adult (9- and 12-week old) Irs2 − / − mice by immunohistochemistry. 9-week old Irs2 − / − mice showed significant thinning of outer retinal layers, concomitant to Muller and microglial cell activation. Photoreceptor cells displayed different signs of degeneration, such as outer/inner segment atrophy, redistribution of rod- and cone-opsins and spatial disorganization of cone cells. This was accompanied by synaptic changes at the outer plexiform layer, including the retraction of rod-spherules, reduction of photoreceptor synaptic ribbons and synaptic remodeling in second order neurons (i.e. loss and sprouting of dendritic processes in rod bipolar and horizontal cells). By 12 weeks of age, the thickness of inner retinal layers was severely affected. Although inner plexiform layer stratification remained unchanged at this stage, rod bipolar cell axon terminals were significantly depleted. Significant loss of Brn3a + retinal ganglion cells occurred in 12-week old Irs2 − / − mice, in contrast to younger ages. Adult Irs2 − / − mice showed clear hallmarks of neurodegeneration and disruption of the inner retina with increasing age. Pharmacological stimulation of Irs2 signaling pathway may provide additional neuroprotection in certain degenerative retinopathies.

12 páginas, 5 figuras.-- et al., Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O2) as somatic cells. We hypothesized that O2 levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O2) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O2 levels; after 24 hours at 5% O2, more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction., This project was supported by the Regenerative Medicine Program of Valencia with funding from the Instituto Carlos III and the Valencian Health Ministry. Other sources of funding include Spanish Ministry for Science and Innovation (MICINN) project SAF2008-00011, Red de Terápia Celular (TerCell), CIBERDEM (ISCIII), and ISCIII project EMER07/012.

9 páginas, 6 figuras.-- El documento en word es la versión post-print., [Aims]: Atherosclerosis is a chronic inflammatory disease regulated by immune mechanisms. CD69 is a cell surface receptor rapidly induced after leukocyte activation at sites of chronic inflammation. Genetic disruption of CD69 in the mouse aggravates collagen-induced arthritis (CIA), and partial depletion of CD69-expressing cells with anti-CD69 monoclonal antibody (mAb) prevents CIA development in wild-type mice, suggesting that this receptor negatively modulates immune and inflammatory responses. It has been recently reported that CD69 is upregulated in a large subset of T cells in atherosclerosis-prone apolipoprotein E-null mice (apoE−/−). In this study, we investigated whether altering CD69 function affects atherosclerosis development. [Methods and results]: We studied native and diet-induced atherosclerosis in apoE−/− and doubly deficient apoE−/−CD69−/− mice and performed expression studies in tissues and primary cells derived from these animals. Plasma cholesterol level was unaffected by CD69 genetic inactivation. Although this genetic manipulation led to an elevated production of interferon γ and interleukin 10 by activated T cells, apoE−/− and apoE−/−CD69−/− mice fed control and high-fat diet exhibited atheromas of similar size and composition when analysed at different stages of the disease. Likewise, anti-CD69 mAb treatment had no effect on plasma cholesterol and atherosclerosis burden in fat-fed apoE−/− mice. [Conclusion]: In contrast to previous studies highlighting the protective function of CD69 against CIA, an autoimmune inflammatory disease, our results rule out a significant role for CD69 against atherosclerosis in apoE−/− mice, an experimental disease model featuring a local inflammatory response triggered and sustained by alterations in lipid homeostasis., Work supported by Instituto de Salud Carlos III—Ministerio de Sanidad y Consumo of Spain (ISCIII-MSC) (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares— RECAVA, and BEFI predoctoral fellowship to S.M.S-G.), Ministerio de Educación y Ciencia of Spain and European Regional Development Fund (grants SAF2004-03057 and SAF2007-62210 to V.A., and BFU2005-08435/BMC to F.S.-M.). M.G. is an investigator of Programa Ramón y Cajal 2002 from Ministerio de Ciencia y Tecnología of Spain and was supported by grant PI06/0937 from ISCIII-MSC.

The definitive version is available at http://www.sciencedirect.com/science/journal/14714914, The tumor suppressor p53 is a transcription factor that is frequently inactivated in human tumors. Therefore,restoring its function has been considered an attractive approach to restrain cancer. Typically, p53-dependent growth arrest, senescence and apoptosis of tumor cells have been attributed to transcriptional activity of nuclear p53. Notably, wild-type p53 gain-of-function enhances cancer resistance in the mouse, but it also accelerates aging in some models, possibly due to altered p53 activity. Therefore, the emerging evidence of mitochondrial transcription-independent activities of p53 has raised high expectations. Here, we review new developments in transcription-dependent and transcription-independent p53 functions, recent advances in targeting p53 for cancer treatment and the pitfalls of moving from the laboratory research to the clinical setting., This Work in the laboratory of V.A. is supported by grants from Ministerio de Sanidad y Consumo/Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares RECAVA), from Sociedad Española de Cardiología, and from Ministerio de Educación y Ciencia and the European Regional Development Fund (SAF2004–03057). Work in the laboratory of U.M.M. is supported by the Nacional Cancer Institute and Philip Morris USA Inc. and Philip Morris International. J.J.F. is supported by a CSIC-I3P program pre-doctoral fellowship cosponsored by the European Social Fund

This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Cardiovascular Research following peer review. The definitive publisher-authenticated version Cardiovasc Res. 75 (4):803-12. is available online at: http://cardiovascres.oxfordjournals.org/cgi/content/full/75/4/803, OBJECTIVE: The tumor suppressor p53 regulates cell proliferation and apoptosis, two key processes in the pathogenesis of occlusive vascular disease. Here, we examined the consequences of heightening p53 function on neointimal lesion formation in the setting of atherosclerosis and mechanical injury. METHODS: (1) Immunohistopathological characterization of neointimal lesions in atherosclerosis-prone apolipoprotein E-null mice with normal p53 gene dosage (apoEKO) and carrying a p53 transgene (Super-p53/apoE-KO); (2) molecular studies in macrophages and smooth muscle cells (SMCs) obtained from these mice. RESULTS: The p53 transgene conferred p53 gain-of-function in cultured cells and mice. In vitro, survival of irradiated Super-p53 macrophages and femoral SMCs was reduced, but only Super-p53 SMCs exhibited attenuated proliferation. In vivo, whereas the size of spontaneously formed and diet-induced aortic atheromas was undistinguishable in apoE-KO and Super-p53/apoE-KO mice, the latter exhibited attenuated neointimal thickening in mechanically-injured femoral artery. In both models, neither apoptosis nor cell proliferation were affected by additional p53 gene dosage when examined in established neointimal lesions. However, at 2 days after mechanical injury when neointimal lesions were not formed yet, cell proliferation was significantly attenuated within medial SMCs of Super-p53/apoEKO mice. CONCLUSION: Heightening p53 function has differential effects on in vitro proliferation of macrophages (unaffected) versus SMCs (reduced), and on native atherosclerosis (unaffected) versus mechanically-induced neointimal thickening (reduced) in apoE-KO mice. The protective effect of p53 in mechanically-injured femoral artery coincided with limited medial SMC proliferation at early time points preceding neointima formation, but neither medial nor neointimal cell proliferation was affected in vessels with established occlusive lesions. These findings corroborate p53 gain-of-function as a promising therapeutic strategy to limit post-angioplasty restenosis but not native atherosclerosis., Work financed by grants from Ministerio de Sanidad y Consumo/Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares, RECAVA), from the Regional Government of Valencia (GV04B-288) and from Ministerio de Educación y Ciencia and the European Regional Development Fund (SAF2004-03057). S.M.S.-G. and J.M.G received salary support from Instituto de Salud Carlos III, and J.J.F. from CSIC-I3P predoctoral fellowship program cosponsored by the European Social Fund.

15 páginas, 8 figuras.-- et al., AP-1 (Activating Protein 1) transcription factor activity is tightly regulated at multiple levels, including dimer formation (i.e., Fos/Jun). Here we show that the intermediate filament protein lamin A/C suppresses AP-1 function through direct interaction with c-Fos, and that both proteins can interact and colocalize at the nuclear envelope (NE) in mammalian cells. Perinuclear localization of c-Fos is absent in Lmna-null cells but can be restored by lamin A overexpression. In vitro, preincubation of c-Fos with lamin A prior to the addition of c-Jun inhibits AP-1 DNA-binding activity. In vivo, overexpression of lamin A reduces the formation of c-Fos/c-Jun heterodimers, and suppresses AP-1 DNA-binding and transcriptional activity. Notably, c-Fos colocalizes with lamin A/C at the NE in starvation-synchronized quiescent cells lacking detectable AP-1 DNA binding. In contrast, serum-induced AP-1 DNA-binding activity coincides with abundant nucleoplasmic c-Fos expression without changes in lamin A/C localization. We also found that Lmna-null cells display enhanced proliferation. In contrast, lamin A overexpression causes growth arrest, and ectopic c-Fos partially overcomes lamin A/C-induced cell cycle alterations. We propose lamin A/C-mediated c-Fos sequestration at the NE as a novel mechanism of transcriptional and cell cycle control., A. Navarro (supported by the Regional Government of Valencia, fellowship CTBFTS/2005/ 036) for technical help with this work; and M.J. Andrés-Manzano for the preparation of figures. This work was supported by grants from Instituto de Salud Carlos III (Red de Centros RECAVA, C03/01), from the Regional Government of Valencia (GV01-488), and from the Spanish Ministry of Education and Science and the European Regional Development Fund (SAF2004-03057). M.K. was supported by an European Union Marie Curie Programme fellowship, S.M.S.-G. by a fellowship of the Instituto de Salud Carlos III, and C.I. by fellowships from the Spanish Ministry of Science and Technology and from the CSIC-I3P Programme.

9 páginas, 4 figuras.-- El documento en word es la versión post-print., Excessive cellular proliferation is thought to contribute to neointimal lesion development during atherosclerosis and restenosis after angioplasty. Inhibition of cyclin-dependent kinase (CDK) activity by p27 inhibits mammalian cell growth. Mounting evidence indicates that p27 negatively regulates neointimal thickening in animal models of restenosis and atherosclerosis, and its expression in human neointimal lesions is consistent with such a protective role. Cell cycle progression is facilitated by cyclinE/CDK2-dependent phosphorylation of p27 on threonine 187 (T187) during late G1. The purpose of this study was to assess whether this phosphorylation event plays a role during atherosclerosis. To this end, we generated apolipoprotein E-null mice with both p27 alleles replaced by a mutated form non-phosphorylatable at T187 (apoE−/−p27T187A mice) and investigated the kinetics of atheroma development in these animals compared to apoE−/− controls with an intact p27 gene. Fat feeding resulted in comparable level of hypercholesterolemia in both groups of mice. Surprisingly, aortic p27 expression was not increased in fat-fed apoE−/−p27T187A mice compared with apoE−/− controls. Moreover, atheroma size, lesion cellularity, proliferation, and apoptotic rates were undistinguishable in both groups of fat-fed mice. Thus, in contrast to previous studies that highlight the importance of p27 phosphorylation at T187 on the control of p27 expression and function in different tissues and pathophysiological scenarios, our findings demonstrate that this phosphorylation event is not implicated in the control of aortic p27 expression and atheroma progression in hypercholesterolemic mice., Work in the laboratory of V.A. is supported inpart by grants fromInstituto de Salud Carlos III (Red de Centros RECAVA, C03/01), from Regional Government of Valencia (GV04B-288), and from Spanish Ministry of Education and Science and Fondo Europeo de DesarrolloRegional(SAF2004-03057).S.M.S.-G. is the recipient of a BEFI Predoctoral fellowship from the Spanish Ministry of Health.

16 páginas, 2 figuras, 1 tabla.-- El documento en word es la versión post-print., Atherosclerosis and associated coronary heart disease events have lower prevalence in women than in men, especially during young adult years. Although multiple lines of evidence suggest that estrogens contribute to this difference, the efficacy of hormone replacement therapy for the prevention of cardiovascular disease in postmenopausal women is controversial. The protective action of estrogen in the cardiovascular system appears to be mediated indirectly by an effect on serum lipoprotein and triglyceride profiles and on the expression of coagulant and fibrinolytic proteins, and by a direct effect on the vessel wall itself. Estrogen has both rapid effects involving alteration of membrane ionic permeability and activation of membrane-bound enzymes and increases in endothelial cell nitric oxide synthase activity, as well as longer-term effects on gene expression that are mediated, at least in part, by the ligand-activated transcription factors, estrogen receptor (alfa) and (beta) . Compounds with pure antiestrogenic activity and selective estrogen receptor modulators that regulate estrogen receptor function in a tissue-specific manner have been developed in an attempt to achieve the cardioprotective effects of estrogens while minimizing the undesirable risks associated with hormone replacement therapy (e.g., endometrial and breast cancer). In this review, we will discuss recent developments on the mechanisms of estrogen action in the cardiovascular system. The results of clinical trials testing the long-term efficacy of hormone replacement therapy for the treatment of cardiovascular disease will also be discussed., Supported in part by grants from the Ministry of Science and Technology of Spain and Fondo Europeo de Desarrollo Regional (grants 1FD97-1035-C02-01 and 02, and SAF2001-2358), Fondo de Investigación Sanitaria (F.I.S.) (grant FIS 01/0917, and Red de Enfermedades Cardiovasculares C03/01), Generalitat de Catalunya, and Human Frontiers Science Program. S. M. Sanz-González received salary support from the F.I.S.

10 páginas.-- El documento en word es la versión post-print., The proteome is described as the entirety of all proteins expressed within a cell at a given moment. In contrast to the stability of the genome, the proteome is highly dynamic and reflects the cell's current status. Since proteins carry out almost all biological functions, the proteome stands in direct relation to cellular functions. Proteomic analysis (i.e., two-dimensional electrophoresis, mass spectrometry and bioinformatics) aims at identifying changes in the composition of the proteome associated to pathophysiologic events that affect basic cellular functions. Functional proteomics expands to understanding the connection between proteomic changes and the state of a cell, taken into account that the observed modifications can be either cause or consequence of the pathological state. Proteomics will not only improve our basic understanding of the factors and molecular mechanisms underlying cardiovascular disease, but also will help identifying novel diagnostic markers and fuel the rational design and discovery of new drugs for medical intervention. This review will discuss basic proteomic approaches relevant to cardiovascular disease, as well as their applications for the identification of biomarkers and drug design.

8 páginas, 6 figuras.-- El docuemnto en word es la versión post-print., Platelet-derived growth factor (PDGF) ligand and receptors (PDGF-R) activate smooth muscle cell (SMC) proliferation, a key event during vascular obstructive disease. The PDGF-R tyrosine kinase inhibitor STI571 attenuates SMC proliferation and experimental neointimal thickening. Here, we investigated the molecular mechanisms underlying STI571-dependent SMC growth arrest. STI571 abrogates PDGF-BB-dependent cyclin D1 and cyclin A protein expression and inhibits transcriptional activation of reporter genes driven by the human cyclin A gene promoter. Repression of cyclin A promoter activity by STI571 requires a functional E2F-binding site, and forced expression of E2F overrides this inhibitory effect. Moreover, STI571 inhibits E2F DNA-binding activity in SMCs. We also found that STI571 abrogates PDGF-BB-dependent activation of extracellular-regulated kinase 1 and 2 (ERK1/2), and forced activation of these factors impaired STI571-dependent inhibition of both cyclin A promoter activity and SMC proliferation. Thus, E2F and ERK1/2 play an important role in STI571-mediated SMC growth arrest and cyclin A transcriptional repression. These findings may have importance in the development of novel therapeutic strategies for the treatment of neointimal hyperplasia., Work in the laboratory of V.A. is supported in part by grants from the Ministerio de Ciencia y Tecnología of Spain and Fondo Europeo de Desarrollo Regional (SAF2001-2358, SAF2002-1443), from Instituto de Salud Carlos III (ISCIII) (Red de Centros C03/01), and from Genera-litat Valenciana (GRUPOS03/072). S.M.S.-G. is a research fellow of the ISCIII. C.C. has been supported by research fellowships from the Agencia Española de Cooperación Internacional and from the ISCIII.

17 páginas.-- El documento en word es la versión post-print., Excessive cell proliferation contributes to the pathobiology of human diseases with a high health and socioeconomic impact, including cancer and vascular occlusive diseases (e.g., atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Recent advances in the understanding of the molecular networks governing the hyperplastic growth of tumors and vascular obstructive neointimal lesions have provided new perspectives for preventive and therapeutic strategies against these disorders. Mammalian cell proliferation requires the activation of several cyclin-dependent protein kinases (CDKs). Postranslational activation of CDKs is a complex process that involves their association with regulatory subunits called cyclins. The activity of CDK / cyclin holoenzymes is negatively regulated through their interaction with members of the CDK family of inhibitory proteins (CKIs). Moreover, over fifty low molecular weight pharmacological CDK inhibitors that target the ATP-binding pocket of the catalytic site of CDKs have been identified. In this review, we will discuss the use of pharmacological and gene therapy strategies against CDK / cyclins in animal models and clinical trials of cancer and cardiovascular disease.

The biological chemistry of nitric oxide (NO) in the oxygenated cellular environment is extremely complex. It involves the direct interaction of NO with specific biomolecules and the so-called indirect effects, due to secondary more potent oxidant species derived from NO which are also able to react with DNA, lipids, thiols and transition metals (Wink et al. 1996; Nathan, 1992). In addition to its regulatory role as a signalling molecule (Nathan, 1992; Moncada and Palmer, 1991) it has become evident that NO (or NO-derived species) is a critical factor involved in various toxicological mechanisms (Wink et al. 1996; Wang et al. 1998; Estevez et al. 1999; Wink et al. 1999). Some controversy exists however about the damaging vs protective actions of NO on oxidative injury, whose biological significance in living cells and tissues remains still ill defined. Research in this laboratory (Lopez-Garcia, 1998; Lopez-Garcia and Sanz-Gonzalez, 2000) has shown that NO synthesis is significantly activated in hepatocytes from control rats following isolation by the classical collagenase-based procedure. NO overproduction appears to be due to the very early activation of liver constitutive Ca2+-dependent NO synthase (cNOS). Previous results have also provided first experimental evidence for the direct involvement of endogenously generated NO as a causal factor responsible for important phenotypic changes commonly observed in short-term cultured hepatocytes, which includes the early impairment of hepatocyte mitochondrial function -i.e., transient cell energy depletion-and glucose metabolism, and the well-known quick and irreversible loss of P450 content (Lopez et al 1987; Lopez-Garcia, 1998). This study aims to further characterise the mechanisms underlying this phenomenon.

10 páginas, 3 figuras.-- El documento en word es la versión post-print., While quiescence is a defining characteristic of differentiated vascular smooth muscle cells (VSMCs) residing within the medial layer of elastic arteries in the adult organism, mature VSMCs can undergo phenotypic modulation and reenter the cell cycle in response to several physiological and pathological stimuli. Abnormal VSMC proliferation is thought to contribute to the pathogenesis of vascular occlusive lesions, including atherosclerosis, vessel renarrowing after successful angioplasty (restenosis), and graft atherosclerosis after coronary transplantation. Therefore, elucidating the molecular mechanisms limiting VSMC growth is currently the subject of active research. This review will focus on the role of cyclin-dependent kinase inhibitory proteins in the regulation of VSMC proliferation and its implication in intimal lesion formation during the pathogenesis of vascular proliferative diseases., Work in the laboratory of V. Andrés is supported in part by grants from the Spanish Dirección General de Educación Superior e Investigación Científica (PM97-0136 and 1FD97-1035-C02-02) and from the American Heart Association, Massachusetts Affiliate (9860022T).