Person:
Crooke Álvarez, Almudena

First Name

Almudena

Last Name

Crooke Álvarez

URI

https://hdl.handle.net/20.500.14352/79888

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Óptica y Optometría

Department

Bioquímica y Biología Molecular

Area

Bioquímica y Biología Molecular

Identifiers

Full item page

Search Results

Now showing 1 - 10 of 21

Melatonin Makes Me Feel Awake! Seven Years of Lab Experience (2000-2007)
(Anales de la Real Academia Nacional de Farmacia, 2007) Pelaez, Teresa; Mediero Muñoz, Aránzazu; Alarma-Estrany, Pilar; Loma Lozano, Patricia; Guzmán Aránguez, Ana Isabel; Crooke Álvarez, Almudena; Colligris, Basilio; Peral Cerda, María Asunción; Pintor Just, Jesús Jerónimo
The research on the effect of melatonin on intraocular pressure (IOP) is reviewed from the hystorical point of view of our laboratory. The original idea of melatonin modulating intraocular pressure has been improved by using selective compounds for MT2 and specially melatonin MT3 receptors. The selective compound 5-methoxyamino N-acetyltryptamine (5-MCA-NAT) has been an attractive compound due to its ability to reduce IOP about 40%, therefore being a good candidate to the treatment of the ocular hypertension linked to glaucoma. More compounds have been developed and tested permitting us to have a more accurate panorama of those receptors controlling the relevant process of intraocular pressure.
Melatonin Receptors Trigger cAMP Production and Inhibit Chloride Movements in Nonpigmented Ciliary Epithelial Cells
(Journal of Pharmacology and Experimental Therapeutics, 2015) Huete Toral, Fernando; Crooke Álvarez, Almudena; Martínez Águila, Alejandro; Pintor Just, Jesús Jerónimo
Melatonin and its analog 5-MCA-NAT (5-methylcarboxyamino-N-acetyl tryptamine) are active compounds reducing intraocular pressure (IOP). This action is mediated through MT2 and the putative MT3 melatonin receptor, producing a transient reduction of IOP that lasts for a few hours and has not yet been characterized. The use of melatonin and its analog are causing a decrease in chloride efflux from rabbit nonpigmented epithelial cells (NPE), possibly explaining the decrease in IOP. Melatonin and 5-MCA-NAT inhibited rabbit NPE chloride release in a concentration-dependent manner, whereas the pD2 values were between 4.5 ± 1.2 and 4.4 ± 1.0, respectively. Melatonin hypotensive action was enhanced by the presence of MT2 antagonists, such as DH97 (N-pentanoyl-2-benzyltryptamine) and 4-P-P-DOT (4-phenyl-2-propionamidotetralin) and by the nonselective melatonin receptor antagonist luzindole. Prazosin (1.5 µM) partially reverses the melatonin action by acting as a selective MT3 antagonist. However, at 15 nM it acts as an α-adrenergic receptor antagonist, enhancing the melatonin effect. Regarding the intracellular pathways triggered by melatonin receptors, neither phospholipase C/protein kinase C pathway nor the canonical reduction of intracellular cAMP was responsible for melatonin or 5-MCA-NAT actions. On the contrary, the application of these substances produced a concentration-dependent increase of cAMP, with pD2 values of 4.6 ± 0.2 and 4.9 ± 0.7 for melatonin and 5-MCA-NAT, respectively. In summary, melatonin reduces the release of chloride concomitantly to cAMP generation. The reduction of Cl− secretion accounts for a decrease in the water outflow and therefore a decrease in aqueous humor production. This could be one of the main mechanisms responsible for the reduction of IOP after application of melatonin and 5-MCA-NAT.
The role and therapeutic potential of melatonin in age-related ocular diseases
(Journal of Pineal Research, 2017) Crooke Álvarez, Almudena; Huete Toral, Fernando; Colligris, Basilio; Pintor Just, Jesús Jerónimo
The eye is continuously exposed to solar UV radiation and pollutants, making it prone to oxidative attacks. In fact, oxidative damage is a major cause of age-related ocular diseases including cataract, glaucoma, age-related macular degeneration and diabetic retinopathy. Since the nature of lens cells, trabecular meshwork cells, retinal ganglion cells, retinal pigment epithelial cells and photoreceptors is post-mitotic, autophagy plays a critical role in their cellular homeostasis. In age-related ocular diseases, this process is impaired, thus, oxidative damage becomes irreversible. Other conditions such as low- grade chronic inflammation and angiogenesis also contribute to the development of retinal diseases (glaucoma, age-related macular degeneration and diabetic retinopathy). As melatonin is known to have remarkable qualities such as antioxidant/antinitridergic, mitochondrial protector, autophagy modulator, anti-inflammatory and anti-angiogenic, it can represent a powerful tool to counteract all these diseases. The present review analyzes the role and therapeutic potential of melatonin in age-related ocular diseases, focusing on nitro-oxidative stress, autophagy, inflammation and angiogenesis mechanisms.
Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study
(Current Eye Research, 2015) Carracedo Rodríguez, Juan Gonzalo; Recchioni, Alberto; Alejandre Alba, Nicolás; Martín Gil, Alba; Crooke Álvarez, Almudena; Jiménez Alfaro-Morote, Ignacio; Pintor Just, Jesús Jerónimo
Purpose: To compare signs and symptoms of dry eye in keratoconus (KC) patients versus healthy subjects. Methods: A total of 15 KC patients (KC group, n = 15 eyes) and 16 healthy subjects (control group, 16 eyes) were enrolled in this study. The Schirmer I test with no anesthetic, tear break-up time (TBUT), corneal staining characteristics, and ocular surface disease index (OSDI) scores were evaluated for both groups. Impression cytology, combined with/scanning laser confocal microscopy (LCM), was performed to evaluate goblet cell density, mucin cloud height (MCH), and goblet cell layer thickness (CLT). Finally, tear concentrations of di-adenosine tetraphosphate (Ap4A) were assessed. Results were statistically analyzed using Shapiro–Wilk and non-parametric Wilcoxon rank sum tests. Statistical significance was set at p < 0.05. Results: KC patients had lower tear volumes and greater corneal staining than did healthy subjects (p < 0.05). OSDI scores were 44.96 ± 8.65 and 17.78 ± 6.50 for the KC and control groups, respectively (p < 0.05). We found no statistically significant differences in TBUT between groups. Impression cytology revealed lower goblet cell densities in KC group patients versus control group subjects (84.88 ± 32.98 and 128.88 ± 50.60 cells/mm,2 respectively, p < 0.05). There was a statistically significant reduction in MCH and CLT in KC group patients compared with control group subjects. Ap4A tear concentrations were higher in KC group patients than in control group subjects (2.56 ± 1.10 and 0.15 ± 0.12 µM, respectively, p < 0.05). Conclusions: The parameters evaluated in this study indicate that KC patients suffer greater symptoms of dry eye and greater tear instability, primarily due to the decreased mucin production in their tears, than do healthy patients with no KC.
Impact of contact lens wear on NLRP3 gene expression: Implications for ocular frailty in middle-aged adults
(Experimental Eye Research, 2020) Crooke Álvarez, Almudena; Martínez Alberquilla, Irene; García Montero, María; Rico Del Viejo, Laura; Ruiz Alcocer, Javier; Madrid Costa, David
The inflammatory process plays a crucial role in frailty syndrome, which can appear in middle age and is associated with a poor health outcome. Consequently, gerontologists recommend screening inflammatory biomarkers in middle-aged adults to detect frailty and, therefore, prevent chronic diseases and mortality. External factors could be a risk factor for frailty because they can generate and extend the inflammatory process. For these reasons, we analysed the effect of long-term contact lens wear on mRNA level of genes linked to inflammation (IL-6, NLRP3, NK1R, CD73, MUC16 and TRPV1 genes) in conjunctival cells of middle-aged individuals, by quantitative PCR. Middle-aged contact lens wearers presented a significant increase of NLRP3 and MUC16 mRNA level as well as a decrease of CD73 mRNA level, in comparison with non-contact lens wearers. Additionally, we checked for a potential correlation between these transcript levels and clinical changes of the participants’ ocular surface. Unlike molecular analysis, clinical examination fails to detect inflammation in contact lens wearers. These data suggest that long-term contact lens wear could trigger an inflammatory response in middle age orchestrated by NLRP3 inflammasome and modulated by CD73 and MUC16 proteins. Further studies are needed to confirm our gene expression findings at the protein level as well as to investigate the potential role of long-term CL wear in the onset of ocular frailty.
Low expression of CD39 and CD73 genes in centenarians compared with octogenarians
(Immunity & Ageing, 2017) Crooke Álvarez, Almudena; Martínez Henández, Juan; Martínez López, Joaquín; Cruz Jentoft, Alfonso; Huete Toral, Fernando; Pintor Just, Jesús Jerónimo
Ageing involves a progressive decline of the body’s regulatory systems including immune system. Adenosine regulates immune function by interaction with its receptors, mainly adenosine A2A receptor, present on the surface of immune cells. Furthermore, cellular response to this nucleoside is highly dependent on its extracellular concentration that is regulated by ecto-enzymes such as CD39 and CD73. Therefore, the aim of this study was to investigate the effect of age on adenosine A2A receptor, CD39 and CD73 gene expression. Changes in mRNA were measured by quantitative PCR from peripheral blood of young, middle-aged and older adults as well as centenarians. Centenarians showed a prominent decrease of CD39 and CD73 mRNA in comparison with older adults. Regarding to adenosine A2A receptor, we detected two subgroups of centenarians with high and low level of transcript. Additionally, adenosine A2A receptor mRNA level of centenarians, did not correlate with their cognitive impairment. In summary, our pilot study suggests that unlike of adenosine A2A receptor, the level of CD39 as well as CD73 mRNA could be a hallmark of successful human ageing.
Therapeutic potential of topical administration of siRNAs against HIF-1α for corneal neovascularization
(Experimental Eye Research, 2022) Peral Cerda, María Asunción; Mateo Ibáñez, Jesús; Domínguez Godínez, Carmen Olalla; Carracedo Rodríguez, Juan Gonzalo; Gómez Pedrero, José Antonio; Crooke Álvarez, Almudena; Pintor Just, Jesús Jerónimo
Given the implications of the problem of neovascularization on ocular health, as well as the growth in the number of cases, the purpose of the present study has been testing the efficacy of siRNAs (small interfering RNA) designed to silence Hypoxia Inducible Factor -1α (HIF-1α) and to demonstrate that their use stops neovascularization in a model of corneal burn. Corneal wounds in the limbic zone were made in the eyes of New Zealand white rabbits. Topical applications of siRNAs were done the next day to the wound for four consecutive days and eyes were examined with a slit lamp. Evaluation of neovascularization progress was done by analyzing images by ImageJTM and to determine the neovascular area in Matlab ® was used. At the same time, a rabbit corneal cell line was used for in vitro study of hypoxia exposure and Western blot analysis of the cell's extracts were done. Under normal cell culture oxygenation, the expression of HIF-1α was lower than that observed under hypoxic conditions. After 2 h of hypoxia, there was a significant increase in the HIF-1α expression, effect that was maintained up to 6 h. The increased in HIF-1α was mimicked by a cell permeable prolyl-4-hydroxylase inhibitor. Cobalt chloride showed no capacity to increase HIF-1α in vitro. The effect of three different siRNA on HIF-1α was tested after 4 h of hypoxia. siRNA#1 was able to silence 80% of HIF-1α expression, siRNA#2 and siRNA#3 reduce the expression in 45% and 40% respectively. In addition, the three siRNA were tested in a corneal model of neovascularization. scrambledsiRNA#2 was the most effective inhibitor of blood vessel production, followed by siRNA#3 and siRNA#1. Compared to the scrambled siRNA (100% of blood vessel generation), siRNA#2 blocked the presence of blood vessels by 83 ± 2%, siRNA#3 inhibited 45 ± 7% and siRNA#1 only inhibited 18 ± 5%. The necessary time to observe the 50% of effect showed values of NV50 of 10.2 ± 2.4 days for the scrambled siRNA, 9.1 ± 1.4 for siRNA#1, 6.5 ± 1.85 for siRNA#2 and 4.8 ± 1.8 days for siRNA#3. In conclusion, the topical application of siRNA towards HIF-1α seems to be an effective and reliable method to stop neovascularization.
Visual function, ocular surface integrity and symptomatology of a new extended depth-of-focus and a conventional multifocal contact lens
(Contact Lens and Anterior Eye, 2020) Martínez Alberquilla, Irene; García Montero, María; Ruiz Alcocer, Javier; Crooke Álvarez, Almudena; Madrid Costa, David
Purpose: To evaluate visual function, ocular surface integrity and dry eye symptoms with an extended depth-of-focus (EDOF) design and a conventional multifocal (MF) contact lens (CL) after 15 days of wear. Methods: A crossover single mask randomised clinical trial was conducted including 30 presbyopes who used an EDOF and a conventional MF CL (Biofinity MF) for 15 days each. Defocus curves, depth-of-focus range, contrast sensitivity (CS) under photopic and mesopic conditions (with and without glare) and subjective perception of halos and glare were evaluated. The ocular surface was evaluated through non-invasive Keratograph tear breakup time (NIKBUT), averaged tear breakup time (NIKBUT-avg), tear meniscus height (TMH), bulbar and limbal redness, and conjunctival and corneal staining. Dry eye symptoms were assessed with the OSDI questionnaire. Results: No statistically significant differences were found for defocus curves or depth-of-focus between the two CLs (both p > 0.05). Subjective perception of halos and glare was not significantly different between CLs. Statistically significant differences were observed for CS under mesopic conditions for low spatial frequencies (p = 0.008). None of the CL produced significant changes in NIKBUT, NIKBUT-avg, TMH or redness. No change in conjunctival staining was observed in 76.7 % and 73.3 % of participants for EDOF and Biofinity MF, respectively. No change in corneal staining was observed in 86.7 % and 83.3 % of participants for EDOF and Biofinity MF, respectively. No changes were observed in the symptomatology measured with OSDI questionnaire (p > 0.05). Conclusions: Both CL for presbyopia offer good visual quality, preserve the ocular surface integrity and provide the patient with similar symptomatology levels after 15 days of lens wear.
An update on dry eye disease molecular treatment: Advances in drug pipelines
(Expert Opinion on Pharmacotherapy, 2014) Colligris, Basilio; Crooke Álvarez, Almudena; Huete Toral, Fernando; Pintor Just, Jesús Jerónimo
Introduction: Dry eye disease is a common disorder provoking changes in tear film and ocular surface. Untreated dry eye could cause ocular infections, corneal ulcer and blindness. Only a few drugs are authorized so far for the treatment of dry eye disease and the possibilities of evolution in this sector are immense. Consequently, a significant number of new potential solutions are under development or placed in the pharmaceutical pipeline, promising better results and lesser side effects. Areas covered: In this article, the corresponding literature and recent Phase III clinical trial data and the corresponding literature, for dry eye disease treatment are reviewed, revealing the new strategic movements in drug pipelines. Expert opinion: From the clinical trial results, the advancement in tear substitutes and secretagogues in addressing specific deficiencies of tear components even though not resolving the underlying conditions of the disease is evident. The vast majority of new compounds under development are anti-inflammatories, steroids, non-steroids and antibiotics; however, there are also some novel lubricating drops and mucin-tear secretagogues. A future aggressive therapy for dry eye, depending on the severity of the symptoms, would include combinations of soft steroids, anti-inflammatories, such as cyclosporine A, with the addition of the new polyvalent mucin and tear secretagogues.
Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways
(Investigative Ophthalmology & Visual Science, 2008) Mediero Muñoz, Aránzazu; Guzmán Aránguez, Ana Isabel; Crooke Álvarez, Almudena; Peral Cerda, María Asunción; Pintor Just, Jesús Jerónimo
Purpose. To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap4A and Ap3A. Methods. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap4A or Ap3A 100 μM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 μM, U0126 100 μM, Y27632 100 nM, and (−)-blebbistatin 10 μM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap4A and Ap3A (100 μM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap4A or Ap3A (100 μM) with U0126 and Y27632 (100 nM). Results. In the presence of Ap4A, U0126, Y27632, AG1478, and (−)-blebbistatin, reduced the migration rate compared to the effect of Ap4A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap4A, respectively). In the presence of Ap3A 100 μM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap3A alone, whereas AG1478 and (−)-blebbistatin (P < 0.0001 versus Ap3A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap4A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. Conclusions. The activation of the Ap4A/P2Y2 receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap3A/P2Y6 receptor signals only the MAPK pathway.