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1.
Int J Mol Sci ; 24(1)2022 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-36613929

RESUMEN

In diabetes, the enzyme arginase is upregulated, which may compete with endothelial nitric oxide (NO) synthase (eNOS) for their common substrate L-arginine and compromise NO-mediated vasodilation. However, this eNOS uncoupling can lead to superoxide production and possibly vasodilator hydrogen peroxide (H2O2) formation to compensate for NO deficiency. This hypothesis was tested in coronary arterioles isolated from pigs with 2-week diabetes after streptozocin injection. The NO-mediated vasodilation induced by flow and VEGF was abolished by NOS inhibitor L-NAME and phosphoinositide 3-kinase (PI3K) inhibitor wortmannin but was not affected by arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) or H2O2 scavenger catalase in control pigs. With diabetes, this vasodilation was partially blunted, and the remaining vasodilation was abolished by catalase and wortmannin. Administration of L-arginine or nor-NOHA restored flow-induced vasodilation in an L-NAME sensitive manner. Diabetes did not alter vascular superoxide dismutase 1, catalase, and glutathione peroxidase mRNA levels. This study demonstrates that endothelium-dependent NO-mediated coronary arteriolar dilation is partially compromised in early type 1 diabetes by reducing eNOS substrate L-arginine via arginase activation. It appears that upregulated arginase contributes to endothelial NO deficiency in early diabetes, but production of H2O2 during PI3K-linked eNOS uncoupling likely compensates for and masks this disturbance.


Asunto(s)
Diabetes Mellitus Tipo 1 , Peróxido de Hidrógeno , Porcinos , Animales , Arteriolas , Peróxido de Hidrógeno/farmacología , Fosfatidilinositol 3-Quinasas , NG-Nitroarginina Metil Éster/farmacología , Arginasa/genética , Catalasa , Factor A de Crecimiento Endotelial Vascular , Fosfatidilinositol 3-Quinasa , Wortmanina/farmacología , Dilatación , Vasos Coronarios , Óxido Nítrico Sintasa de Tipo III/genética , Vasodilatación , Arginina/farmacología , Endotelio Vascular
2.
Int J Mol Sci ; 22(18)2021 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-34575925

RESUMEN

Protein kinase C (PKC) activation can evoke vasoconstriction and contribute to coronary disease. However, it is unclear whether PKC activation, without activating the contractile machinery, can lead to coronary arteriolar dysfunction. The vasoconstriction induced by the PKC activator phorbol 12,13-dibutyrate (PDBu) was examined in isolated porcine coronary arterioles. The PDBu-evoked vasoconstriction was sensitive to a broad-spectrum PKC inhibitor but not affected by inhibiting PKCß2 or Rho kinase. After exposure of the vessels to a sub-vasomotor concentration of PDBu (1 nmol/L, 60 min), the endothelium-dependent nitric oxide (NO)-mediated dilations in response to serotonin and adenosine were compromised but the dilation induced by the NO donor sodium nitroprusside was unaltered. PDBu elevated superoxide production, which was blocked by the superoxide scavenger Tempol. The impaired NO-mediated vasodilations were reversed by Tempol or inhibition of PKCß2, xanthine oxidase, c-Jun N-terminal kinase (JNK) and Rho kinase but were not affected by a hydrogen peroxide scavenger or inhibitors of NAD(P)H oxidase and p38 kinase. The PKCß2 protein was detected in the arteriolar wall and co-localized with endothelial NO synthase. In conclusion, activation of PKCß2 appears to compromise NO-mediated vasodilation via Rho kinase-mediated JNK signaling and superoxide production from xanthine oxidase, independent of the activation of the smooth muscle contractile machinery.


Asunto(s)
Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , Óxido Nítrico/metabolismo , Proteína Quinasa C beta/metabolismo , Vasodilatación , Animales , Inmunohistoquímica , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C beta/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Porcinos , Vasodilatación/genética , Vasodilatadores/farmacología , Xantina Oxidasa/metabolismo
3.
J Mol Cell Cardiol ; 131: 82-90, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31015037

RESUMEN

Diabetes is associated with cardiac inflammation and impaired endothelium-dependent coronary vasodilation, but molecular mechanisms involved in this dysfunction remain unclear. We examined contributions of inflammatory molecules lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), stress-activated kinases (c-Jun N-terminal kinase [JNK] and p38), arginase, and reactive oxygen species to coronary arteriolar dysfunction in a porcine model of type 1 diabetes. Coronary arterioles were isolated from streptozocin-induced diabetic pigs and control pigs for vasoreactivity and molecular/biochemical studies. Endothelium-dependent nitric oxide (NO)-mediated vasodilation to serotonin was diminished after 2 weeks of diabetes, without altering endothelium-independent vasodilation to sodium nitroprusside. Superoxide scavenger TEMPOL, NO precursor L-arginine, arginase inhibitor nor-NOHA, anti-LOX-1 antibody or JNK inhibitors SP600125 and BI-78D3 improved dilation of diabetic vessels to serotonin. However, hydrogen peroxide scavenger catalase, anti-IgG antibody or p38 kinase inhibitor SB203580 had no effect. Combined inhibition of arginase and superoxide levels did not further improve vasodilation. Arginase-I mRNA expression, LOX-1 and JNK protein expression, and superoxide levels were elevated in diabetic arterioles. In conclusion, sequential activation of LOX-1, JNK, and L-arginine consuming enzyme arginase-I in diabetes elicits superoxide-dependent oxidative stress and impairs endothelial NO-mediated dilation in coronary arterioles. Therapeutic targeting of these adverse vascular molecules may improve coronary arteriolar function during diabetes.


Asunto(s)
Arginasa/metabolismo , Arteriolas/metabolismo , Vasos Coronarios/metabolismo , Diabetes Mellitus Experimental/complicaciones , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Receptores Depuradores de Clase E/metabolismo , Animales , Antracenos/farmacología , Arginasa/genética , Arteriolas/patología , Vasos Coronarios/patología , Óxidos N-Cíclicos/farmacología , Diabetes Mellitus Experimental/metabolismo , Dioxanos/farmacología , Imidazoles/farmacología , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Masculino , Óxido Nítrico/farmacología , Piridinas/farmacología , Receptores Depuradores de Clase E/genética , Serotonina/farmacología , Marcadores de Spin , Porcinos , Tiazoles/farmacología , Vasodilatación/efectos de los fármacos
4.
Am J Pathol ; 188(3): 818-827, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29309745

RESUMEN

Hypertension is associated with numerous diseases, but its direct impact on the ocular circulation and neuroretinal function remains unclear. Herein, mouse eyes were challenged with different levels of hemodynamic insult via transverse aortic coarctation, which increased blood pressure and flow velocity by 50% and 40%, respectively, in the right common carotid artery, and reduced those parameters by 30% and 40%, respectively, in the left common carotid artery. Blood velocity in the right central retinal artery gradually increased up to 40% at 4 weeks of transverse aortic coarctation, and the velocity in the left central retinal artery gradually decreased by 20%. The fundus and retinal architecture were unaltered by hemodynamic changes. Endothelium-dependent vasodilations to acetylcholine and adenosine were reduced only in right (hypertensive) ophthalmic arteries. Increased cellularity in the nerve fiber/ganglion cell layers, enhanced glial fibrillary acidic protein expression, and elevated superoxide level were found only in hypertensive retinas. The electroretinogram showed decreased scotopic b-waves in the hypertensive eyes and decreased scotopic oscillatory potentials in both hypertensive and hypotensive eyes. In conclusion, hypertension sustained for 4 weeks causes ophthalmic vascular dysfunction, retinal glial cell activation, oxidative stress, and neuroretinal impairment. Although ophthalmic vasoregulation is insensitive to hypotensive insult, the ocular hypoperfusion causes neuroretinal dysfunction.


Asunto(s)
Arteria Oftálmica/fisiopatología , Retina/fisiopatología , Vasos Retinianos/fisiopatología , Animales , Velocidad del Flujo Sanguíneo/fisiología , Presión Sanguínea/fisiología , Electrorretinografía , Hemodinámica/fisiología , Masculino , Ratones , Flujo Sanguíneo Regional/fisiología
5.
Exp Eye Res ; 177: 181-190, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30120928

RESUMEN

Spectral domain optical coherence tomography (SD-OCT) is used as a non-invasive tool for retinal morphological assessment in vivo. Information on the correlation of SD-OCT with retinal histology in the porcine retina, a model resembling the human retina, is limited. Herein, we correlated the hypo- and hyper-reflective bands on SD-OCT with histology of the lamellar architecture and cellular constituents of the porcine retina. SD-OCT images were acquired with the Heidelberg Spectralis HRA + OCT. Histological analysis was performed using epoxy resin embedded tissue and transmission electron microscopy. Photomicrographs from the histologic sections were linearly scaled to correct for tissue shrinkage and correlated with SD-OCT images. SD-OCT images correlated well with histomorphometric data. A hyper-reflective band in the mid-to-outer inner nuclear layer correlated with the presence of abundant mitochondria in horizontal cell processes and adjacent bipolar cells. A concentration of cone nuclei corresponded to a relative hypo-reflective band in the outer portion of the outer nuclear layer. The presence of 3 hyper-reflective bands in the outer retina corresponded to: 1) the external limiting membrane; 2) the cone and rod ellipsoid zones; and 3) the interdigitation zone of photoreceptor outer segments/retinal pigment epithelium (RPE) apical cell processes and the RPE. These correlative and normative SD-OCT data may be employed to characterize and assess the in vivo histologic changes in retinal vascular and degenerative diseases and the responses to novel therapeutic interventions in this large animal model.


Asunto(s)
Técnicas Histológicas , Microscopía Electrónica , Imagen Óptica/métodos , Retina/anatomía & histología , Tomografía de Coherencia Óptica/métodos , Animales , Porcinos
6.
Circ Res ; 114(1): 92-100, 2014 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-24141169

RESUMEN

RATIONALE: Studies in cultured endothelium implicate that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) or Fcγ receptor II (CD32) contributes to the proatherogenic effects of C-reactive protein (CRP). However, the identity of the receptors linking to deleterious actions of CRP in vasomotor regulation remains unknown. OBJECTIVE: We tested the hypothesis that LOX-1 contributes to adverse effects of CRP on endothelium-dependent vasomotor function in resistance arterioles. METHODS AND RESULTS: Porcine coronary arterioles were isolated for vasoreactivity study, dihydroethidium fluorescence staining of superoxide, immunohistochemical localization of receptors, immunoprecipitation of receptor/CRP interaction, and protein blot. Intraluminal treatment of pressurized arterioles with a pathophysiological level of CRP (7 µg/mL; 60 minutes) attenuated endothelium-dependent nitric oxide-mediated and prostacyclin-mediated dilations to serotonin and arachidonic acid, respectively. LOX-1 and CD32 were detected in the endothelium of arterioles. Blockade of LOX-1 with either pharmacological antagonist κ-carrageenan or anti-LOX-1 antibody prevented the detrimental effect of CRP on vasodilator function, whereas anti-CD32 antibody treatment was ineffective. Denudation of endothelium and blockade of LOX-1 but not CD32 prevented CRP-induced elevation of superoxide in the vessel wall. CRP was coimmunoprecipitated with LOX-1 and CD32 from CRP-treated arterioles. Similarly, LOX-1 and CD32 blockade prevented CRP-induced arteriolar expression of plasminogen activator inhibitor-1, a thrombogenic protein. CONCLUSIONS: CRP elicits endothelium-dependent oxidative stress and compromises nitric oxide-mediated and prostacyclin-mediated vasomotor function via LOX-1 activation. In contrast, both LOX-1 and CD32 mediate plasminogen activator inhibitor-1 upregulation in arterioles by CRP. Thus, activation of LOX-1 and CD32 may contribute to vasomotor dysfunction and proatherogenic actions of CRP, respectively.


Asunto(s)
Proteína C-Reactiva/farmacología , Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , Receptores Depuradores de Clase E/metabolismo , Vasodilatación , Animales , Arteriolas/metabolismo , Arteriolas/fisiopatología , Carragenina/farmacología , Línea Celular , Vasos Coronarios/fisiopatología , Endotelio Vascular/efectos de los fármacos , Epoprostenol/farmacología , Humanos , Técnicas In Vitro , Óxido Nítrico/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores Depuradores de Clase E/antagonistas & inhibidores , Receptores Depuradores de Clase E/genética , Superóxidos/metabolismo , Porcinos
7.
J Mol Cell Cardiol ; 86: 75-84, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26211713

RESUMEN

Elevated levels of endothelin-1 (ET-1), a potent vasoactive peptide, are implicated as a risk factor for cardiovascular diseases by exerting vasoconstriction. The aim of this study was to address whether ET-1, at sub-vasomotor concentrations, elicits adverse effects on coronary microvascular function. Porcine coronary arterioles (50-100µm) were isolated, cannulated and pressurized without flow for in vitro study. Diameter changes were recorded using a videomicrometer. Arterioles developed basal tone (60±3µm) and dilated to the endothelium-dependent nitric oxide (NO)-mediated vasodilators serotonin (1nmol/L to 0.1µmol/L) and adenosine (1nmol/L to 10µmol/L). Treating the vessels with a clinically relevant sub-vasomotor concentration of ET-1 (10pmol/L, 60min) significantly attenuated arteriolar dilations to adenosine and serotonin but not to endothelium-independent vasodilator sodium nitroprusside. The arteriolar wall contains ETA receptors and the adverse effect of ET-1 was prevented by ETA receptor antagonist BQ123, the superoxide scavenger Tempol, the NADPH oxidase inhibitors apocynin and VAS2870, the NOX2-based NADPH oxidase inhibitor gp91 ds-tat, or the p38 kinase inhibitor SB203580. However, ETB receptor antagonist BQ788, H2O2 scavenger catalase, scrambled gp91 ds-tat, or inhibitors of xanthine oxidase (allopurinol), PKC (Gö 6983), Rho kinase (Y27632), and c-Jun N-terminal kinase (SP600125) did not protect the vessel. Immunohistochemical staining showed that ET-1 elicited Tempol-, apocynin- and SB203580-sensitive superoxide productions in the arteriolar wall. Our results indicate that exposure of coronary arterioles to a pathophysiological, sub-vasomotor concentration of ET-1 leads to vascular dysfunction by impairing endothelium-dependent NO-mediated dilation via p38 kinase-mediated production of superoxide from NADPH oxidase following ETA receptor activation.


Asunto(s)
Enfermedades Cardiovasculares/genética , Vasos Coronarios/metabolismo , Endotelina-1/metabolismo , NADPH Oxidasas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenosina/administración & dosificación , Animales , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Arteriolas/patología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Vasos Coronarios/patología , Endotelina-1/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Peróxido de Hidrógeno/metabolismo , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Nitroprusiato/administración & dosificación , Serotonina/administración & dosificación , Porcinos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Vasodilatación/efectos de los fármacos , Vasodilatación/genética , Vasodilatadores/administración & dosificación , Proteínas Quinasas p38 Activadas por Mitógenos/genética
8.
J Biol Chem ; 289(42): 29446-56, 2014 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-25190815

RESUMEN

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.


Asunto(s)
Canales de Calcio/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/fisiología , Factores de Transcripción NFATC/fisiología , Proteínas de Neoplasias/fisiología , Calcio/metabolismo , Silenciador del Gen , Histamina/química , Humanos , Inflamación , Interleucina-8/metabolismo , Interleucinas/metabolismo , Proteína ORAI1 , Proteína ORAI2 , Interferencia de ARN , Transducción de Señal , Molécula de Interacción Estromal 1
9.
J Biol Chem ; 288(16): 11263-72, 2013 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-23447534

RESUMEN

The entry of extracellular Ca(2+), which is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels, is essential for T cell activation and the normal functioning of other immune cells. Although the molecular components of CRAC channels, the Orai1 pore-forming subunit and the STIM1-activating subunit have been recently identified, the gating mechanism by which Orai1 channels conduct Ca(2+) entry upon Orai1-STIM1 interaction following Ca(2+) store release remains elusive. Herein, we show that C-terminal truncations or point mutations prevented Orai1 from binding to STIM1 and subsequent channel opening. In contrast, an Orai1 mutant with an N-terminal truncation interacted with but failed to be activated by STIM1. Moreover, Orai1 channels with C-terminal disruption, but not N-terminal truncation, could be gated by fused functional domains of STIM1. Interestingly, the channel activities of Orai1 mutants carrying either an N-terminal or a C-terminal truncation were restored by a methionine mutation at the putative gating hinge, the conserved Gly-98 site in the first transmembrane segment (TM1) of Orai1. Collectively, these results support a stepwise gating mechanism of STIM1-operated Orai1 channels; the initial binding between STIM1 and the C terminus of Orai1 docks STIM1 onto the N terminus of Orai1 to initiate conformational changes of the pore-lining TM1 helix of Orai1, leading to the opening of the channel.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canales de Calcio/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Mutación , Proteínas de Neoplasias/genética , Proteína ORAI1 , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Molécula de Interacción Estromal 1
10.
Exp Eye Res ; 103: 63-70, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22940370

RESUMEN

The purpose of this study was to investigate the roles of endothelium-derived factors in the retinal arteriolar responses to acute severe elevation in systemic blood pressure (BP) in cats. Acute elevation of mean arterial BP by 60% for 5 min was achieved by inflating a balloon-tipped catheter in the descending aorta. The retinal arteriolar diameter, flow velocity, wall shear rate (WSR) and blood flow (RBF) changes during BP elevation were assessed with laser Doppler velocimetry 2 h after intravitreal injections of nitric oxide (NO) synthase inhibitor l-NAME, cyclooxygenase inhibitor indomethacin, endothelin-1 receptor antagonists (BQ-123 for type A and BQ-788 for type B), or Rho kinase inhibitor fasudil. BP elevation caused a marked increase in retinal arteriolar flow velocity and WSR with slight vasoconstriction, resulting in an increase in RBF. The increases in velocity, WSR and RBF, but not diameter, were correlated with the increase in ocular perfusion pressure. With l-NAME or indomethacin, the increase in RBF upon BP elevation was significantly attenuated due to enhanced retinal arteriolar vasoconstriction. In contrast, BQ-123 and fasudil potentiated the increased RBF. BQ-788 had no effect on arteriolar diameter and hemodynamics. Our data suggest that acute elevation of BP by 60% leads to an increase in RBF due to the release of NO and prostanoids probably through a shear stress-induced vasodilation mechanism. The release of endothelin-1 and Rho kinase activation help to limit RBF augmentation by counteracting the vasodilation. It appears that the retinal endothelium, by releasing vasoactive substances, contributes to RBF regulation during acute severe elevation of systemic blood pressure.


Asunto(s)
Presión Sanguínea/fisiología , Endotelio Vascular/fisiología , Hipertensión/fisiopatología , Arteria Retiniana/fisiología , Animales , Antihipertensivos/administración & dosificación , Arteriolas/fisiología , Velocidad del Flujo Sanguíneo/fisiología , Gatos , Inhibidores de la Ciclooxigenasa/administración & dosificación , Endotelina-1/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Femenino , Frecuencia Cardíaca/fisiología , Presión Intraocular/fisiología , Inyecciones Intravítreas , Flujometría por Láser-Doppler , Masculino , NG-Nitroarginina Metil Éster/administración & dosificación , Óxido Nítrico/metabolismo , Arteria Retiniana/efectos de los fármacos , Vasodilatadores/administración & dosificación , Quinasas Asociadas a rho/metabolismo
11.
Am J Ophthalmol ; 239: 230-243, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35307380

RESUMEN

PURPOSE: To investigate the effect of stanniocalcin-1 (STC-1), a secreted polypeptide exhibiting multiple functions in cell survival and death, on photoreceptor degeneration in a porcine model of retinitis pigmentosa (RP). METHODS: P23H transgenic pigs (TG P23H) and wild-type hybrid littermates were obtained from the National Swine Resource and Research Center. Human recombinant STC-1 was injected intravitreally every 2 weeks from postnatal day 15 (P15) to P75. The contralateral eye was injected with balanced salt solution as a control. Electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed to evaluate retinal function and morphology in vivo at P90. Retinal tissue was collected for histologic analysis and molecular assays to evaluate the antioxidative and anti-inflammatory mechanisms by which STC-1 may rescue photoreceptor degeneration. RESULTS: Intravitreal injection of STC-1 improved retinal function in TG P23H pigs with increased photopic and flicker ERG a- and b-wave amplitudes. Greater integrity of the ellipsoid zone (EZ) band on SD-OCT and morphologic rescue with preservation of cone photoreceptors were observed in STC-1-treated TG P23H pigs. STC-1 altered gene expression in TG P23H pig retina on microarray analysis and increased photoreceptor specific gene expression by reverse transcription-polymerase chain reaction analysis. STC-1 significantly decreased oxidative stress and the expressions of NLRP3 inflammasome, cleaved caspase-1, and IL-1ß in TG P23H pig retina. CONCLUSIONS: Intravitreal administration of STC-1 enhances cone photoreceptor function, improves EZ integrity, and reduces retinal degeneration through antioxidative and anti-inflammatory effects in a large animal (pig) model of the most common form of autosomal dominant RP in the United States.


Asunto(s)
Degeneración Retiniana , Retinitis Pigmentosa , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Glicoproteínas , Humanos , Inflamación , Estrés Oxidativo , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/genética , Degeneración Retiniana/prevención & control , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/genética , Porcinos
12.
Am J Physiol Heart Circ Physiol ; 301(3): H683-8, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21666123

RESUMEN

In subtotal nephrectomy (SN)- and salt-induced hypertension, calcitonin gene-related peptide (CGRP) plays a compensatory role to attenuate the blood pressure increase in the absence of an increase in the neuronal synthesis and release of this peptide. Therefore, the purpose of this study was to determine whether the mechanism of this antihypertensive activity is through enhanced sensitivity of the vasculature to the dilator actions of this neuropeptide. Hypertension was induced in Sprague-Dawley rats by SN and 1% saline drinking water. Control rats were sham-operated and given tap water to drink. After 11 days, rats had intravenous (drug administration) and arterial (continuous mean arterial pressure recording) catheters surgically placed and were studied in a conscious unrestrained state. Baseline mean arterial pressure was higher in the SN-salt rats (157 ± 5 mmHg) compared with controls (128 ± 3 mmHg). Administration of CGRP (and adrenomedullin) produced a significantly greater dose-dependent decrease in mean arterial pressure in SN-salt rats compared with controls (∼2.0-fold for both the low and high doses). Interestingly, isolated superior mesenteric arterioles from SN-salt rats were significantly more responsive to the dilator effects of CGRP (but not adenomedullin) than the controls (pEC(50), SN-salt, 14.0 ± 0.1 vs. control, 12.0 ± 0.1). Analysis of the CGRP receptor proteins showed that only the receptor component protein was increased significantly in arterioles from SN-salt rats. These data indicate that the compensatory antihypertensive effects of CGRP result from an increased sensitivity of the vasculature to dilator activity of this peptide. The mechanism may be via the upregulation of receptor component protein, thereby providing a more efficient coupling of the receptor to the signal transduction pathways.


Asunto(s)
Antihipertensivos/administración & dosificación , Péptido Relacionado con Gen de Calcitonina/administración & dosificación , Hipertensión/tratamiento farmacológico , Mesenterio/irrigación sanguínea , Nefrectomía , Cloruro de Sodio Dietético , Vasodilatación/efectos de los fármacos , Vasodilatadores/administración & dosificación , Adrenomedulina/administración & dosificación , Análisis de Varianza , Animales , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Arteriolas/fisiopatología , Presión Sanguínea/efectos de los fármacos , Western Blotting , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipertensión/etiología , Hipertensión/metabolismo , Hipertensión/fisiopatología , Infusiones Intravenosas , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Péptido Relacionado con el Gen de Calcitonina/agonistas , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Factores de Tiempo
13.
Microcirculation ; 18(1): 36-45, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21166924

RESUMEN

Oxidized low-density lipoprotein (OxLDL) causes impairment of endothelium-dependent, nitric oxide (NO)-mediated vasodilation involving l-arginine deficiency. However, the underlying mechanism remains elusive. Since arginase and endothelial NO synthase (eNOS) share the substrate l-arginine, we hypothesized that OxLDL may reduce l-arginine availability to eNOS for NO production, and thus vasodilation, by up-regulating arginase. To test this hypothesis, porcine subepicardial arterioles (70-130 µm) were isolated for vasomotor study and for immunohistochemical detection of arginase and eNOS expressions. The coronary arterioles dilated dose-dependently to the endothelium-dependent NO-mediated vasodilator serotonin. This vasodilation was inhibited in the same manner by NOS inhibitor N(G)-nitro-l-arginine methyl ester and by lumenal OxLDL (0.5 mg protein/mL). The inhibitory effect of OxLDL was reversed after treating the vessels with either l-arginine (3 mM) or arginase inhibitor difluoromethylornithine (DFMO; 0.4 mM). Consistent with vasomotor alterations, OxLDL inhibited serotonin-induced NO release from coronary arterioles and this inhibition was reversed by DFMO. Vascular arginase activity was significantly elevated by OxLDL. Immunohistochemical analysis indicated that OxLDL increased arginase I expression in the vascular wall without altering eNOS expression. Taken together, these results suggest that OxLDL up-regulates arginase I, which contributes to endothelial dysfunction by reducing l-arginine availability to eNOS for NO production and thus vasodilation.


Asunto(s)
Arginasa/biosíntesis , Arginina/metabolismo , Vasos Coronarios/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico/metabolismo , Vasodilatación/fisiología , Animales , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Porcinos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Vasodilatación/efectos de los fármacos
14.
Microvasc Res ; 82(3): 356-63, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21983453

RESUMEN

Endothelium-derived hyperpolarizing factor (EDHF) is an important vasodilator that regulates the vasomotor function. However, it remains unclear whether diabetes/hyperglycemia-induced vascular impairments extend to the EDHF. The present study aims to determine the effect of high glucose (HG) on EDHF-mediated arteriolar dilation and the underlying mechanism. Porcine coronary arterioles were isolated and pressurized for vasomotor study. Cultured porcine coronary artery endothelial cells (ECs) were used for molecular and biochemical analysis. Our results demonstrate that bradykinin (BK)-simulated arteriolar dilation is mediated by nitric oxide (NO) and EDHF pathways. Direct incubation of HG impaired vasodilation to BK but not to sodium nitroprusside (endothelium-independent vasodilator). In the presence of inhibitors of endothelial NO synthase (eNOS) and cyclooxygenase, the EDHF-mediated dilation was reduced by HG incubation. The inhibitory effect of HG was prevented by treating the vessels with superoxide scavenger Tempol. In cultured coronary endothelial cells, HG reduced endothelial epoxyeicosatrienoic acid (EET) production as well as cytochrome P450 epoxygenase (CYP) activity. Furthermore, the superoxide production was elevated in ECs after HG incubation. Pretreatment with Tempol before HG incubation prevented the increase of cellular superoxide and abolished the decrease of CYP activity. Collectively, our results suggest that, in addition to NO-mediated pathway, HG impairs the EET/EDHF-mediated vasodilation in coronary arterioles via the elevated level of superoxide leading to inhibition of CYP activity in coronary ECs.


Asunto(s)
Factores Biológicos/metabolismo , Vasos Coronarios/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Glucosa/metabolismo , Hiperglucemia/enzimología , Vasodilatación , Animales , Arteriolas/enzimología , Arteriolas/fisiopatología , Células Cultivadas , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiopatología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Eicosanoides/metabolismo , Células Endoteliales/enzimología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Hiperglucemia/fisiopatología , Técnicas In Vitro , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Porcinos , Factores de Tiempo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología
15.
Diabetes ; 70(10): 2353-2363, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34353852

RESUMEN

Diabetes elevates endothelin-1 (ET-1) in the vitreous and enhances constriction of retinal venules to this peptide. However, mechanisms contributing to ET-1-induced constriction of retinal venules are incompletely understood. We examined roles of sodium-hydrogen exchanger 1 (NHE1), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and extracellular calcium (Ca2+) in retinal venular constriction to ET-1 and the impact of diabetes on these signaling molecules. Retinal venules were isolated from control pigs and pigs with streptozocin-induced diabetes for in vitro studies. ET-1-induced vasoconstriction was abolished in the absence of extracellular Ca2+ and sensitive to c-Jun N-terminal kinase (JNK) inhibitor SP600125 but unaffected by extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 kinase inhibitor SB203580, or broad-spectrum PKC inhibitor Gö 6983. Diabetes (after 2 weeks) enhanced venular constriction to ET-1, which was insensitive to PD98059 and Gö 6983 but was prevented by NHE1 inhibitor cariporide, SB203580, and SP600125. In conclusion, extracellular Ca2+ entry and activation of JNK, independent of ERK and PKC, mediate constriction of retinal venules to ET-1. Diabetes activates p38 MAPK and NHE1, which cause enhanced venular constriction to ET-1. Treatments targeting these vascular molecules may lessen retinal complications in early diabetes.


Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Endotelina-1/farmacología , Vena Retiniana , Intercambiador 1 de Sodio-Hidrógeno/fisiología , Vasoconstricción , Animales , Calcio/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/metabolismo , Angiopatías Diabéticas/fisiopatología , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/fisiopatología , Endotelina-1/sangre , Endotelina-1/fisiología , Imidazoles/farmacología , Masculino , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/fisiología , Piridinas/farmacología , Vena Retiniana/efectos de los fármacos , Vena Retiniana/metabolismo , Vena Retiniana/fisiopatología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Intercambiador 1 de Sodio-Hidrógeno/genética , Porcinos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
16.
Methods Mol Biol ; 2319: 77-85, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34331245

RESUMEN

The laser-induced choroidal neovascularization (CNV) model has been widely used for research on wet age-related macular degeneration (wet-AMD) and other ocular neovascular diseases. In this model, the Bruch membrane is perforated by laser injury, resulting in neovascularization formed from the choroidal capillaries. It has become a standard method to evaluate the effect of different treatments on CNV progression in preclinical studies. This protocol can be used in various species, including rat, mouse, pig, and monkey. The rodent laser-induced CNV model is the most commonly used because of the advantages in both cost- and time-efficiency. It takes only 10-15 min to complete the whole laser procedure after adequate training and practicing the technique. Peak CNV formation occurs at approximately 2 weeks after laser application. The entire protocol may require up to 3 weeks to complete the treatment, fundus image acquisition, and tissue collection for histologic analysis. This chapter describes the detailed procedures, protocols, and useful notes on how to induce CNV by laser.


Asunto(s)
Neovascularización Coroidal , Degeneración Macular , Anestesia , Animales , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Rayos Láser , Degeneración Macular/patología , Ratas
17.
Methods Mol Biol ; 2319: 111-117, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34331249

RESUMEN

The retina offers a unique opportunity to directly visualize blood vessels in vivo noninvasively. Over the past few decades, several new imaging techniques have been adapted to study the retinal vasculature in the laboratory in animal models and in the clinic in human subjects. High-contrast, finely detailed fundus images can be acquired by confocal scanning laser ophthalmoscopy (cSLO). With fluorescein angiography (FA), the retinal microcirculation can be visualized. High-resolution spectral-domain optical coherence tomography (SD-OCT) is able to acquire cross-section images resolving the microarchitecture of the retina, similar to histology. The techniques and protocols for acquiring cSLO, FA, and SD-OCT imaging of the retinal vasculature and morphology in the rodent are described.


Asunto(s)
Angiografía con Fluoresceína/métodos , Oftalmoscopía/métodos , Retina/diagnóstico por imagen , Vasos Retinianos/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodos , Animales , Angiografía con Fluoresceína/instrumentación , Retina/metabolismo , Vasos Retinianos/metabolismo , Tomografía de Coherencia Óptica/instrumentación
18.
Sci Rep ; 11(1): 18401, 2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34526573

RESUMEN

We investigated and compared the susceptibility of retinal blood flow regulation and neural function in mice developing type 2 diabetes. The longitudinal changes in retinal neuronal function and blood flow responses to a 10-min systemic hyperoxia and a 3-min flicker stimulation were evaluated every 2 weeks in diabetic db/db mice and nondiabetic controls (db/m) from age 8 to 20 weeks. The retinal blood flow and neural activity were assessed using laser speckle flowgraphy and electroretinography (ERG), respectively. The db/db mice had significantly higher blood glucose levels and body weight. The resting retinal blood flow was steady and comparable between two groups throughout the study. Hyperoxia elicited a consistent decrease, and flicker light an increase, in retinal blood flow in db/m mice independent of age. However, these flow responses were significantly diminished in db/db mice at 8 weeks old and then the mice became unresponsive to stimulations at 12 weeks. Subsequently, the ERG implicit time for oscillatory potential was significantly increased at 14 weeks of age while the a-wave and b-wave amplitudes and implicit times remained unchanged. The deficiencies of flow regulation and neurovascular coupling in the retina appear to precede neural dysfunction in the mouse with type 2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Flujo Sanguíneo Regional , Neuronas Retinianas/metabolismo , Vasos Retinianos/fisiopatología , Animales , Biomarcadores , Retinopatía Diabética/etiología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Electrorretinografía , Hipoxia/metabolismo , Ratones , Neuronas Retinianas/patología
19.
Sci Rep ; 10(1): 19796, 2020 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-33188259

RESUMEN

This study aimed to evaluate longitudinal changes in retinal blood flow in response to flicker stimulation and systemic hyperoxia in mice using a laser speckle flowgraphy (LSFG-Micro). The retinal blood flow in vascular area surrounding the optic nerve head was measured in 8-week-old male mice every 2 weeks until age 20-week. The coefficient of variation of retinal blood flow under resting condition was analyzed every 2 weeks to validate the consistency of the measurement. On day 1 of the experiment, retinal blood flow was assessed every 20 s for 6 min during and after 3 min flicker light (12 Hz) stimulation; on day 2, retinal blood flow was measured every minute for 20 min during and after 10 min systemic hyperoxia; and on day 3, electroretinography (ERG) was performed. Body weight, systemic blood pressure, and ocular perfusion pressure increased significantly with age, but the resting retinal blood flow and ERG parameters remained unchanged. Retinal blood flow significantly increased with flicker stimulation and decreased with systemic hyperoxia, independent of age. The LSFG-Micro provides consistent and reproducible retinal blood flow measurement in adult mice. Longitudinal assessments of retinal blood flow in response to flicker stimulation and systemic hyperoxia may be useful indexes for noninvasive monitoring of vascular function in retinas.


Asunto(s)
Hiperoxia/fisiopatología , Animales , Velocidad del Flujo Sanguíneo , Presión Sanguínea/fisiología , Electrorretinografía , Hemodinámica/fisiología , Flujometría por Láser-Doppler , Masculino , Ratones , Ratones Endogámicos C57BL , Vasos Retinianos/fisiología
20.
Invest Ophthalmol Vis Sci ; 61(5): 36, 2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32437549

RESUMEN

Purpose: Retinal vasomotor activity can be regulated by two major endothelial enzymes, nitric oxide synthase (NOS) and cyclooxygenase (COX). The vascular arginase also consumes a NOS substrate and thus impedes NOS-mediated vasodilation. Diabetes mellitus exhibits vascular complications in the retina with elevated oxidative stress and compromised NOS-mediated vasodilation. However, the underlying molecular mechanisms remain unclear, and the effect of diabetes on COX-mediated vasodilation is unknown. Herein, we examined the relative impact of diabetes on retinal arteriolar dilations to COX and NOS activation and the roles of arginase and superoxide in diabetes-induced vasomotor dysfunction. Methods: Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks of hyperglycemia, 433 ± 27 mg/dL) or age-matched control pigs (97 ± 4 mg/dL). The vasodilations to bradykinin (NOS activator) and histamine (NOS/COX activator) were examined in vitro. Results: Retinal arteriolar dilations to histamine and bradykinin were significantly reduced after 2 weeks of diabetes. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated the dilations of control vessels, but not diabetic vessels, to histamine. In the presence of L-NAME and COX inhibitor indomethacin, histamine-induced dilations of control and diabetic vessels were reduced similarly. Treatment of diabetic vessels with arginase inhibitor nor-NOHA, but not superoxide dismutase mimetic TEMPOL, preserved both histamine- and bradykinin-induced dilations in an L-NAME-sensitive manner. Conclusions: Arginase, rather than superoxide, impairs endothelium-dependent NOS-mediated dilation of retinal arterioles during diabetes, whereas vasodilation mediated by COX remains intact. Blockade of vascular arginase may improve endothelial function of retinal arterioles during early onset of diabetes.


Asunto(s)
Arginasa/fisiología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Endotelio Vascular/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Retiniana/fisiología , Vasodilatación/fisiología , Animales , Arteriolas/fisiología , Glucemia/metabolismo , Bradiquinina/farmacología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/enzimología , Inhibidores Enzimáticos/farmacología , Histamina/farmacología , Hiperglucemia/fisiopatología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sus scrofa
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