NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-β, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS

Influencia del peroxinitrito en los cambios vasculares observados tras la isquemia cerebral focal en rata

Evidencias experimentales demuestran que tras un episodio transitorio de isquemia cerebral focal se inducen cambios estructurales, mecánicos y miogénicos en la pared de las arterias cerebrales, así como un aumento en la producción de peroxinitrito, tanto en los vasos sanguíneos como en el parénquima cerebral. Todas estas alteraciones se han asociado con la expansión del daño cerebral. El peroxinitrito es una especie oxidante/nitrante que, debido a su elevada capacidad de modificar proteínas, puede deteriorar diferentes tejidos, incluido el cerebro. De acuerdo con ello, la presente tesis doctoral hemos hipotetizado que el peroxinitrito es un factor clave implicado en las alteraciones de la arteria cerebral media y del daño cerebral que se observan tras episodios de isquemia-reperfusión. El ácido úrico es un potente antioxidante endógeno que supone las 2/3 partes del poder antioxidante del plasma en humanos, y que tras su administración exógena ha mostrado efectos protectores alentadores en la isquemia. Sin embargo, hasta la fecha, únicamente un estudio ha mostrado una reducción del volumen de infarto cerebral y del daño neurológico después del tratamiento con ácido úrico administrado en un plazo clínicamente relevante. Por otro lado, en estudios previos, el desarrollo de hiperemia reactiva durante el periodo de reperfusión después de un episodio de isquemia cerebral se asoció a un mayor volumen de infarto y déficit neurológico. En la presente tesis confirmamos que las ratas hiperémicas sufren infartos más severos que las que no presentan hiperemia, y que el ácido úrico administrado a los 135 min después de la oclusión de la arteria cerebral media es capaz de atenuar dicho infarto y el daño neurológico asociado. Por lo tanto, demostramos que el ácido úrico administrado exógenamente es altamente efectivo en ratas que presentan signos de hiperemia durante la reperfusión, es decir, en aquellas ratas que padecen un mayor daño cerebral. Además, mostramos que la vasorrelajación inducida por SLIGRL, agonista de los receptores PAR2, en arteria cerebral media precontraída con U46619, análogo estable del TXA2 y agonista de los receptores TP, es mayor en ratas isquémicas que en sham (control). También demostramos que este incremento de la relajación tras la isquemia-referfusión podría estar relacionado con la incapacidad del U46619 para bloquear la relajación mediada por los canales SKCa endoteliales. El mecanismo subyacente a esta observación implicaría un aumento de la acumulación de peroxinitrito y la consiguiente disrupción de la F-actina endotelial, que limitaría la capacidad del U46619 para inhibir la función de los canales SKCa, facilitando así la relajación por EDH tras un episodio de isquemia-referfusión. En conjunto, los resultados obtenidos en el presente estudio muestran que, tras la isquemia-referfusión, el peroxinitrito, mediante su actividad oxidante/nitrosilante, participa en las alteraciones estructurales, mecánicas y vasodilatadoras que se observan en la arteria cerebral media tras un episodio de I/R. Estos hallazgos ayudan a comprender la fisiopatología del ictus isquémico, a la vez que refuerzan el potencial terapéutico de los compuestos antioxidantes con capacidad de neutralizar la acción del peroxinitrito.Experimental evidence shows that, after a transient episode of focal cerebral ischemia, structural, mechanical and myogenic changes in the wall of the cerebral arteries, as well as an increased production of peroxynitrite, are induced in both blood vessels and brain parenchyma. All these alterations have been associated with the expansion of brain damage. Peroxynitrite is an oxidizing/nitriding specie that due to its high capacity to modify proteins, can deteriorate different tissues, including the brain. Accordingly, in this thesis we hypothesized that peroxynitrite is a key factor involved in the alterations of the middle cerebral artery and brain damage observed after episodes of ischemia-reperfusion. Uric acid is a powerful antioxidant endogenous that represents 2/3 of plasma antioxidant in humans and that after exogenous administration has shown promising protective effects in ischemia. However, to date, only one study has shown a reduction in brain infarct volume and neurological damage after treatment with uric acid administered within clinically relevant time. Furthermore, in previous studies, the development of reactive hyperemia during the reperfusion period after a cerebral ischemic episode was associated with increased infarct volume and neurological deficit. In this thesis we confirm that the hyperemic rats suffer more severe infarcts than those ones that don’t experiment hyperemia, and uric acid administered to 135 min after occlusion of the middle cerebral artery is able to attenuate said infarction and associated neurological damage. Therefore, we show that exogenously administered uric acid is highly effective in rats signs of hyperemia during reperfusion, namely, in those rats having higher brain damage. Moreover, we show that vasorelaxation induced by SLIGRL, PAR2 receptor agonist, in middle cerebral artery precontracted with U46619, stable analog of TXA2 and TP receptors agonist, is higher in ischemic rats than in controls (sham). Furthermore, we demonstrate that this increase in relaxation after ischemia-reperfusion may be related to the inability of U46619 to block endothelial relaxation mediated by SKCa channels. The mechanism underlying this observation involve increased accumulation peroxynitrite and consequent disruption of F-actin endothelial, which limit the ability of U46619 to inhibit the function of SKCa channels, thus facilitating the relaxation after an episode of ischemia-reperfusion. Overall, the results obtained in this study show that, after ischemia-reperfusion, the peroxynitrite participates in the structural, mechanical and vasodilator alterations observed in the middle cerebral artery. These findings help to understand the pathophysiology of ischemic stroke, while reinforcing the therapeutic potential of antioxidant compounds capable of neutralizing the action of peroxynitrite

Ligands exert biased activity to regulate sigma 1 receptor interactions with cationic TRPA1, TRPV1, and TRPM8 channels

The sigma 1 receptor (σ1R) and the mu-opioid receptor (MOR) regulate the transient receptor potential (TRP) V1 calcium channel. A series of proteins are involved in the cross-regulation between MORs and calcium channels like the glutamate N-methyl-D-aspartate receptor (NMDAR), including the histidine triad nucleotide-binding protein 1 (HINT1), calmodulin (CaM), and the σ1R. Thus, we assessed whether similar mechanisms also apply to the neural TRP ankyrin member 1 (TRPA1), TRP vanilloid member 1 (TRPV1), and TRP melastatin member 8 (TRPM8). Our results indicate that σ1R and CaM bound directly to cytosolic regions of these TRPs, and this binding increased in the presence of calcium. By contrast, the association of HINT1 with these TRPs was moderately dependent on calcium. The σ1R always competed with CaM for binding to the TRPs, except for its binding to the TRPA1 C-terminal where σ1R binding cooperated with that of CaM. However, σ1R dampened HINT1 binding to the TRPA1 N-terminal. When the effect of σ1R ligands was addressed, the σ1R agonists PRE084 and pregnenolone sulfate enhanced the association of the σ1R with the TRPM8 N-terminal and TRPV1 C-terminal in the presence of physiological calcium, as seen for the σ1R–NMDAR interactions. However, these agonists dampened σ1R binding to the TRPA1 and TRPV1 N-terminal domains, and also to the TRPA1 C-terminal, as seen for σ1R–binding immunoglobulin protein (BiP) interactions in the endoplasmic reticulum (ER). By contrast, the σ1R antagonists progesterone and S1RA reduced the association of σ1R with TRPA1 and TRPV1 C-terminal regions, as seen for the σ1R–NMDAR interactions. Conversely, they enhanced the σ1R interaction with the TRPA1 N-terminal, as seen for σ1R–BiP interactions, whereas they barely affected the association of σ1R with the TRPV1 N-terminal. Thus, depending on the calcium channel and the cytosolic region examined, the σ1R agonists pregnenolone sulfate and PRE084 opposed or collaborated with the σ1R antagonists progesterone and S1RA to disrupt or promote such interactions. Through the use of cloned cytosolic regions of selected TRP calcium channels, we were able to demonstrate that σ1R ligands exhibit biased activity to regulate particular σ1R interactions with other proteins. Since σ1Rs are implicated in essential physiological processes, exploiting such ligand biases may represent a means to develop more selective and efficacious pharmacological interventions.This work was supported by MICINN Plan Nacional I+D+i [grant number RT 2018-093677-B-100]. EC-M was supported by a Grant from MECD [FPU 15/02356]

Middle cerebral artery remodeling following transient brain ischemia is linked to early postischemic hyperemia: A target of uric acid treatment

© 2015 the American Physiological Society. Ischemia impairs blood supply to the brain, and reperfusion is important to restore cerebral blood flow (CBF) and rescue neurons from cell death. However, reperfusion can induce CBF values exceeding the basal values before ischemia. This hyperemic effect has been associated with a worse ischemic brain damage, albeit the mechanisms that contribute to infarct expansion are not clear. In this study, we investigated the influence of early postischemic hyperemia on brain damage and middle cerebral artery (MCA) properties and the effect of treatment with the endogenous antioxidant uric acid (UA). The MCA was occluded for 90 min followed by 24 h reperfusion in adult male Sprague-Dawley rats. Cortical CBF increases at reperfusion beyond 20% of basal values were taken as indicative of hyperemia. UA (16 mg/kg) or vehicle (Locke's buffer) was administered intravenously 135 min after MCA occlusion. Hyperemic compared with nonhyperemic rats showed MCA wall thickening (sham: 22.4 ± 0.8 μm; nonhyperemic: 23.1 ± 1.2 μm; hyperemic: 27.8 ± 0.9 at 60 mmHg; P < 0.001, hyperemic vs. sham) involving adventitial cell proliferation, increased oxidative stress, and interleukin-18, and more severe brain damage. Thus MCA remodeling after ischemia-reperfusion takes place under vascular oxidative and inflammatory stress conditions linked to hyperemia. UA administration attenuated MCA wall thickening, induced passive lumen expansion, and reduced brain damage in hyperemic rats, although it did not increase brain UA concentration. We conclude that hyperemia at reperfusion following brain ischemia induces vascular damage that can be attenuated by administration of the endogenous antioxidant UA.Peer Reviewe

Cannabidiol enhances morphine antinociception, diminishes NMDA-mediated seizures and reduces stroke damage via the sigma 1 receptor

Cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, exhibits therapeutic potential for various human diseases, including chronic neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, ischemic stroke, epilepsy and other convulsive syndromes, neuropsychiatric disorders, neuropathic allodynia and certain types of cancer. CBD does not bind directly to endocannabinoid receptors 1 and 2, and despite research efforts, its specific targets remain to be fully identified. Notably, sigma 1 receptor (σ1R) antagonists inhibit glutamate N-methyl-D-aspartate acid receptor (NMDAR) activity and display positive effects on most of the aforesaid diseases. Thus, we investigated the effects of CBD on three animal models in which NMDAR overactivity plays a critical role: opioid analgesia attenuation, NMDA-induced convulsive syndrome and ischemic stroke. In an in vitro assay, CBD disrupted the regulatory association of σ1R with the NR1 subunit of NMDAR, an effect shared by σ1R antagonists, such as BD1063 and progesterone, and prevented by σ1R agonists, such as 4-IBP, PPCC and PRE084. The in vivo administration of CBD or BD1063 enhanced morphine-evoked supraspinal antinociception, alleviated NMDA-induced convulsive syndrome, and reduced the infarct size caused by permanent unilateral middle cerebral artery occlusion. These positive effects of CBD were reduced by the σ1R agonists PRE084 and PPCC, and absent in σ1R−/− mice. Thus, CBD displays antagonist-like activity toward σ1R to reduce the negative effects of NMDAR overactivity in the abovementioned experimental situations.This work was supported by MINECO Plan Nacional I + D + i [grant number SAF-2015-65420R].Peer reviewe

The σ1 Receptor and the HINT1 Protein Control α2δ1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain

Nerve injury produces neuropathic pain through the binding of α2δ1 proteins to glutamate N-methyl-D-aspartate receptors (NMDARs). Notably, mice with a targeted deletion of the sigma 1 receptor (σ1R) gene do not develop neuropathy, whereas mice lacking the histidine triad nucleotide-binding protein 1 (Hint1) gene exhibit exacerbated allodynia. σ1R antagonists more effectively diminish neuropathic pain of spinal origin when administered by intracerebroventricular injection than systemically. Thus, in mice subjected to unilateral sciatic nerve chronic constriction injury (CCI), we studied the participation of σ1Rs and HINT1 proteins in the formation of α2δ1-NMDAR complexes within the supraspinal periaqueductal gray (PAG). We found that δ1 peptides required σ1Rs in order to interact with the NMDAR NR1 variant that contains the cytosolic C1 segment. σ1R antagonists or low calcium levels provoke the dissociation of σ1R-NR1 C1 dimers, while they barely affect the integrity of δ1-σ1R-NR1 C1 trimers. However, HINT1 does remove δ1 peptides from the trimer, thereby facilitating the subsequent dissociation of σ1Rs from NMDARs. In σ1R−/− mice, CCI does not promote the formation of NMDAR-α2δ1 complexes and allodynia does not develop. The levels of α2δ1-σ1R-NMDAR complexes increase in HINT1−/− mice and after inducing CCI, degradation of α2δ1 proteins is observed. Notably, σ1R antagonists but not gabapentinoids alleviate neuropathic pain in these mice. During severe neuropathy, the metabolism of α2δ1 proteins may account for the failure of many patients to respond to gabapentinoids. Therefore, σ1Rs promote and HINT1 proteins hinder the formation α2δ1-NMDAR complexes in the PAG, and hence, the appearance of mechanical allodynia depends on the interplay between these proteins

Influencia del peroxinitrito en los cambios vasculares observados tras la isquemia cerebral focal en rata

Evidencias experimentales demuestran que tras un episodio transitorio de isquemia cerebral focal se inducen cambios estructurales, mecánicos y miogénicos en la pared de las arterias cerebrales, así como un aumento en la producción de peroxinitrito, tanto en los vasos sanguíneos como en el parénquima cerebral. Todas estas alteraciones se han asociado con la expansión del daño cerebral. El peroxinitrito es una especie oxidante/nitrante que, debido a su elevada capacidad de modificar proteínas, puede deteriorar diferentes tejidos, incluido el cerebro. De acuerdo con ello, la presente tesis doctoral hemos hipotetizado que el peroxinitrito es un factor clave implicado en las alteraciones de la arteria cerebral media y del daño cerebral que se observan tras episodios de isquemia-reperfusión. El ácido úrico es un potente antioxidante endógeno que supone las 2/3 partes del poder antioxidante del plasma en humanos, y que tras su administración exógena ha mostrado efectos protectores alentadores en la isquemia. Sin embargo, hasta la fecha, únicamente un estudio ha mostrado una reducción del volumen de infarto cerebral y del daño neurológico después del tratamiento con ácido úrico administrado en un plazo clínicamente relevante. Por otro lado, en estudios previos, el desarrollo de hiperemia reactiva durante el periodo de reperfusión después de un episodio de isquemia cerebral se asoció a un mayor volumen de infarto y déficit neurológico. En la presente tesis confirmamos que las ratas hiperémicas sufren infartos más severos que las que no presentan hiperemia, y que el ácido úrico administrado a los 135 min después de la oclusión de la arteria cerebral media es capaz de atenuar dicho infarto y el daño neurológico asociado. Por lo tanto, demostramos que el ácido úrico administrado exógenamente es altamente efectivo en ratas que presentan signos de hiperemia durante la reperfusión, es decir, en aquellas ratas que padecen un mayor daño cerebral. Además, mostramos que la vasorrelajación inducida por SLIGRL, agonista de los receptores PAR2, en arteria cerebral media precontraída con U46619, análogo estable del TXA2 y agonista de los receptores TP, es mayor en ratas isquémicas que en sham (control). También demostramos que este incremento de la relajación tras la isquemia-referfusión podría estar relacionado con la incapacidad del U46619 para bloquear la relajación mediada por los canales SKCa endoteliales. El mecanismo subyacente a esta observación implicaría un aumento de la acumulación de peroxinitrito y la consiguiente disrupción de la F-actina endotelial, que limitaría la capacidad del U46619 para inhibir la función de los canales SKCa, facilitando así la relajación por EDH tras un episodio de isquemia-referfusión. En conjunto, los resultados obtenidos en el presente estudio muestran que, tras la isquemia-referfusión, el peroxinitrito, mediante su actividad oxidante/nitrosilante, participa en las alteraciones estructurales, mecánicas y vasodilatadoras que se observan en la arteria cerebral media tras un episodio de I/R. Estos hallazgos ayudan a comprender la fisiopatología del ictus isquémico, a la vez que refuerzan el potencial terapéutico de los compuestos antioxidantes con capacidad de neutralizar la acción del peroxinitrito.Experimental evidence shows that, after a transient episode of focal cerebral ischemia, structural, mechanical and myogenic changes in the wall of the cerebral arteries, as well as an increased production of peroxynitrite, are induced in both blood vessels and brain parenchyma. All these alterations have been associated with the expansion of brain damage. Peroxynitrite is an oxidizing/nitriding specie that due to its high capacity to modify proteins, can deteriorate different tissues, including the brain. Accordingly, in this thesis we hypothesized that peroxynitrite is a key factor involved in the alterations of the middle cerebral artery and brain damage observed after episodes of ischemia-reperfusion. Uric acid is a powerful antioxidant endogenous that represents 2/3 of plasma antioxidant in humans and that after exogenous administration has shown promising protective effects in ischemia. However, to date, only one study has shown a reduction in brain infarct volume and neurological damage after treatment with uric acid administered within clinically relevant time. Furthermore, in previous studies, the development of reactive hyperemia during the reperfusion period after a cerebral ischemic episode was associated with increased infarct volume and neurological deficit. In this thesis we confirm that the hyperemic rats suffer more severe infarcts than those ones that don't experiment hyperemia, and uric acid administered to 135 min after occlusion of the middle cerebral artery is able to attenuate said infarction and associated neurological damage. Therefore, we show that exogenously administered uric acid is highly effective in rats signs of hyperemia during reperfusion, namely, in those rats having higher brain damage. Moreover, we show that vasorelaxation induced by SLIGRL, PAR2 receptor agonist, in middle cerebral artery precontracted with U46619, stable analog of TXA2 and TP receptors agonist, is higher in ischemic rats than in controls (sham). Furthermore, we demonstrate that this increase in relaxation after ischemia-reperfusion may be related to the inability of U46619 to block endothelial relaxation mediated by SKCa channels. The mechanism underlying this observation involve increased accumulation peroxynitrite and consequent disruption of F-actin endothelial, which limit the ability of U46619 to inhibit the function of SKCa channels, thus facilitating the relaxation after an episode of ischemia-reperfusion. Overall, the results obtained in this study show that, after ischemia-reperfusion, the peroxynitrite participates in the structural, mechanical and vasodilator alterations observed in the middle cerebral artery. These findings help to understand the pathophysiology of ischemic stroke, while reinforcing the therapeutic potential of antioxidant compounds capable of neutralizing the action of peroxynitrite

Peroxynitrite formed during a transient episode of brain ischemia increases endothelium-derived hyperpolarization-type dilations in thromboxane/prostaglandin receptor-stimulated rat cerebral arteries

AIM: Increased thromboxane A2 and peroxynitrite are hallmarks of cerebral ischaemia/reperfusion (I/R). Stimulation of thromboxane/prostaglandin receptors (TP) attenuates endothelium-derived hyperpolarization (EDH). We investigated whether EDH-type middle cerebral artery (MCA) relaxations following TP stimulation are altered after I/R and the influence of peroxynitrite. METHODS: Vascular function was determined by wire myography after TP stimulation with the thromboxane A2 mimetic 9,11-dideoxy-9α, 11α -methano-epoxy prostaglandin F2α (U46619) in MCA of Sprague Dawley rats subjected to MCA occlusion (90 min)/reperfusion (24 h) or sham operation, and in non-operated (control) rats. Some rats were treated with saline or the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (20 mg kg-1 ). Protein expression was evaluated in MCA and in human microvascular endothelial cells submitted to hypoxia (overnight)/reoxygenation (24 h) (H/R) using immunofluorescence and immunoblotting. RESULTS: In U46619-pre-constricted MCA, EDH-type relaxation by the proteinase-activated receptor 2 agonist serine-leucine-isoleucine-glycine-arginine-leucine-NH2 (SLIGRL) was greater in I/R than sham rats due to an increased contribution of small-conductance calcium-activated potassium channels (SKCa ), which was confirmed by the enlarged relaxation to the SKCa activator N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine. I/R and H/R induced endothelial protein tyrosine nitration and filamentous-actin disruption. In control MCA, either cytochalasin D or peroxynitrite disrupted endothelial filamentous-actin and augmented EDH-type relaxation. Furthermore, peroxynitrite decomposition during I/R prevented the increase in EDH-type responses. CONCLUSION: Following TP stimulation in MCA, EDH-type relaxation to SLIGRL is greater after I/R due to endothelial filamentous-actin disruption by peroxynitrite, which prevents TP-induced block of SKCa input to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote post-ischaemic brain injury

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-β, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS