RESUMEN
50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human ß 3-adrenoreceptor (Tgß 3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgß 3 rats. The aortic expression of ⢠NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate ⢠NO and O2 â¢- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgß 3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgß 3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tgß 3: 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tgß 3: 1.55 ± 0.17; p < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgß 3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tgß 3: 0.13 ± 0.05; p < 0.05). Aortic â¢NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgß 3 rats (+52% vs. WT). Aortic O2 â¢- production was increased in Tgß 3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.
Asunto(s)
Insuficiencia Cardíaca , Enfermedades Vasculares , Animales , Aorta/metabolismo , Insuficiencia Cardíaca/metabolismo , Humanos , Ratas , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil-treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.
Asunto(s)
Miembro Posterior , Isquemia , Óxido Nítrico Sintasa de Tipo III , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Citrato de Sildenafil , Animales , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Miembro Posterior/metabolismo , Isquemia/tratamiento farmacológico , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal , Citrato de Sildenafil/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Intracranial aneurysm (IA) is a frequent and generally asymptomatic cerebrovascular abnormality characterized as a localized dilation and wall thinning of intracranial arteries that preferentially arises at the arterial bifurcations of the circle of Willis. The devastating complication of IA is its rupture, which results in subarachnoid hemorrhage that can lead to severe disability and death. IA affects about 3% of the general population with an average age for detection of rupture around 50 years. IAs, whether ruptured or unruptured, are more common in women than in men by about 60% overall, and more especially after the menopause where the risk is double-compared to men. Although these data support a protective role of estrogen, differences in the location and number of IAs observed in women and men under the age of 50 suggest that other underlying mechanisms participate to the greater IA prevalence in women. The aim of this review is to provide a comprehensive overview of the current data from both clinical and basic research and a synthesis of the proposed mechanisms that may explain why women are more prone to develop IA.
RESUMEN
OBJECTIVE: CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), caused by dominant mutations in the NOTCH3 receptor, is the most aggressive small vessel disease of the brain. A key feature of its pathogenesis is accumulation of the extracellular domain of NOTCH3 receptor (Notch3ECD ) in small vessels, with formation of characteristic extracellular deposits termed granular osmiophilic material (GOM). Here, we investigated the therapeutic potential of a mouse monoclonal antibody (5E1) that specifically recognizes Notch3ECD . METHODS: The binding affinity of 5E1 toward purified NOTCH3 was assessed using Octet analysis. The ability of 5E1 to bind Notch3ECD deposits in brain vessels and its effects on disease-related phenotypes were evaluated in the CADASIL mouse model, which overexpresses a mutant rat NOTCH3. Notch3ECD and GOM deposition, white matter lesions, and cerebral blood flow deficits were assessed at treatment initiation (10 weeks) and study completion (30 weeks) using quantitative immunohistochemistry, electron microscopy, and laser-Doppler flowmetry. RESULTS: 5E1 antibody bound recombinant rat NOTCH3 with an average affinity of 317nM. A single peripheral injection of 5E1 robustly decorated Notch3ECD deposits in the brain vasculature. Chronic administration of 5E1 did not attenuate Notch3ECD or GOM deposition and was not associated with perivascular microglial activation. It also failed to halt the development of white matter lesions. Despite this, 5E1 treatment markedly protected against impaired cerebral blood flow responses to neural activity and topical application of vasodilators and normalized myogenic responses of cerebral arteries. INTERPRETATION: This study establishes immunotherapy targeting Notch3ECD as a new avenue for disease-modifying treatment in CADASIL that warrants further development. Ann Neurol 2018;84:246-259.
Asunto(s)
CADASIL/metabolismo , CADASIL/terapia , Circulación Cerebrovascular/fisiología , Matriz Extracelular/metabolismo , Inmunoterapia/métodos , Receptor Notch3/metabolismo , Animales , CADASIL/inmunología , Matriz Extracelular/inmunología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Unión Proteica/fisiología , Ratas , Receptor Notch3/administración & dosificación , Receptor Notch3/inmunologíaRESUMEN
Intracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)-which encodes a circulating pro-angiogenic factor mainly secreted from the liver-shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA.
Asunto(s)
Proteínas Similares a la Angiopoyetina/genética , Predisposición Genética a la Enfermedad , Aneurisma Intracraneal/genética , Mutación/genética , Sistemas de Lectura Abierta/genética , Proteína 6 similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/sangre , Células Cultivadas , Codón sin Sentido/genética , Familia , Femenino , Células HEK293 , Humanos , Aneurisma Intracraneal/sangre , Masculino , Persona de Mediana Edad , Linaje , Factores de RiesgoRESUMEN
Abnormal activity of the renin-angiotensin-aldosterone system plays a causal role in the development of hypertension, atherosclerosis, and associated cardiovascular events such as myocardial infarction, stroke, and heart failure. As both a vasoconstrictor and a proinflammatory mediator, angiotensin II (Ang II) is considered a potential link between hypertension and atherosclerosis. However, a role for Ang II-induced inflammation in atherosclerosis has not been clearly established, and the molecular mechanisms and intracellular signaling pathways involved are not known. Here, we demonstrated that the RhoA GEF Arhgef1 is essential for Ang II-induced inflammation. Specifically, we showed that deletion of Arhgef1 in a murine model prevents Ang II-induced integrin activation in leukocytes, thereby preventing Ang II-induced recruitment of leukocytes to the endothelium. Mice lacking both LDL receptor (LDLR) and Arhgef1 were protected from high-fat diet-induced atherosclerosis. Moreover, reconstitution of Ldlr-/- mice with Arhgef1-deficient BM prevented high-fat diet-induced atherosclerosis, while reconstitution of Ldlr-/- Arhgef1-/- with WT BM exacerbated atherosclerotic lesion formation, supporting Arhgef1 activation in leukocytes as causal in the development of atherosclerosis. Thus, our data highlight the importance of Arhgef1 in cardiovascular disease and suggest targeting Arhgef1 as a potential therapeutic strategy against atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Leucocitos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Vasculitis/metabolismo , Angiotensina II/genética , Angiotensina II/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Inflamación/genética , Inflamación/mortalidad , Inflamación/patología , Leucocitos/patología , Ratones , Ratones Noqueados , Receptores de LDL/deficiencia , Factores de Intercambio de Guanina Nucleótido Rho/genética , Vasculitis/genética , Vasculitis/patologíaRESUMEN
CADASIL (Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy), the most frequent genetic cause of stroke and vascular dementia, is caused by highly stereotyped mutations in the NOTCH3 receptor, which is predominantly expressed in vascular smooth muscle. The well-established TgNotch3R169C mouse model develops characteristic features of the human disease, with deposition of NOTCH3 and other proteins, including TIMP3 (tissue inhibitor of metalloproteinase 3), on brain vessels, as well as reduced maximal dilation, and attenuated myogenic tone of cerebral arteries, but without elevated blood pressure. Increased TIMP3 levels were recently shown to be a major determinant of altered myogenic tone. In this study, we investigated the contribution of TIMP3 and Notch3 signaling to the impairment of maximal vasodilator capacity caused by the archetypal R169C mutation. Maximally dilated cerebral arteries in TgNotch3R169C mice exhibited a decrease in lumen diameter over a range of physiological pressures that occurred before myogenic tone deficits. This defect was not prevented by genetic reduction of TIMP3 in TgNotch3R169C mice and was not observed in mice overexpressing TIMP3. Knock-in mice with the R169C mutation (Notch3R170C/R170C) exhibited similar reductions in arterial lumen, and both TgNotch3R169C and Notch3R170C/R170C mice showed increased cerebral artery expression of Notch3 target genes. Reduced maximal vasodilation was prevented by conditional reduction of Notch activity in smooth muscle of TgNotch3R169C mice and mimicked by conditional activation of Notch3 in smooth muscle, an effect that was blood pressure-independent. We conclude that increased Notch3 activity mediates reduction in maximal dilator capacity of cerebral arteries in CADASIL and may contribute to reductions in cerebral blood flow.
Asunto(s)
Arterias Cerebrales/metabolismo , Arterias Cerebrales/patología , Trastornos Cerebrovasculares/genética , ADN/genética , Mutación , Receptor Notch3/genética , Vasodilatación , Animales , Arterias Cerebrales/fisiopatología , Circulación Cerebrovascular , Trastornos Cerebrovasculares/metabolismo , Trastornos Cerebrovasculares/fisiopatología , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Receptor Notch3/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cerebral small vessel disease (SVD) is a leading cause of stroke and dementia. CADASIL, an inherited SVD, alters cerebral artery function, compromising blood flow to the working brain. TIMP3 (tissue inhibitor of metalloproteinase 3) accumulation in the vascular extracellular matrix in CADASIL is a key contributor to cerebrovascular dysfunction. However, the linkage between elevated TIMP3 and compromised cerebral blood flow (CBF) remains unknown. Here, we show that TIMP3 acts through inhibition of the metalloprotease ADAM17 and HB-EGF to regulate cerebral arterial tone and blood flow responses. In a clinically relevant CADASIL mouse model, we show that exogenous ADAM17 or HB-EGF restores cerebral arterial tone and blood flow responses, and identify upregulated voltage-dependent potassium channel (KV) number in cerebral arterial myocytes as a heretofore-unrecognized downstream effector of TIMP3-induced deficits. These results support the concept that the balance of TIMP3 and ADAM17 activity modulates CBF through regulation of myocyte KV channel number.
Asunto(s)
Proteína ADAM17/metabolismo , Encéfalo/fisiología , CADASIL/fisiopatología , Hemodinámica , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
OBJECTIVE: CADASIL is a genetic paradigm of cerebral small vessel disease caused by NOTCH3 mutations that stereotypically lead to the extracellular deposition of NOTCH3 ectodomain (Notch3(ECD) ) on the vessels. TIMP3 and vitronectin are 2 extracellular matrix proteins that abnormally accumulate in Notch3(ECD) -containing deposits on brain vessels of mice and patients with CADASIL. Herein, we investigated whether increased levels of TIMP3 and vitronectin are responsible for aspects of CADASIL disease phenotypes. METHODS: Timp3 and vitronectin expression were genetically reduced in TgNotch3(R169C) mice, a well-established preclinical model of CADASIL. A mouse overexpressing human TIMP3 (TgBAC-TIMP3) was developed. Disease-related phenotypes, including cerebral blood flow (CBF) deficits, white matter lesions, and Notch3(ECD) deposition, were evaluated between 6 and 20 months of age. RESULTS: CBF responses to neural activity (functional hyperemia), topical application of vasodilators, and decreases in blood pressure (CBF autoregulation) were similarly reduced in TgNotch3(R169C) and TgBAC-TIMP3 mice, and myogenic responses of brain arteries were likewise attenuated. These defects were rescued in TgNotch3(R169C) mice by haploinsufficiency of Timp3, although the number of white matter lesions was unaffected. In contrast, haploinsufficiency or loss of vitronectin in TgNotch3(R169C) mice ameliorated white matter lesions, although CBF responses were unchanged. Amelioration of cerebrovascular reactivity or white matter lesions in these mice was not associated with reduced Notch3(ECD) deposition in brain vessels. INTERPRETATION: Elevated levels of TIMP3 and vitronectin, acting downstream of Notch3(ECD) deposition, play a role in CADASIL, producing divergent influences on early CBF deficits and later white matter lesions.
Asunto(s)
Encéfalo/patología , CADASIL/patología , CADASIL/fisiopatología , Circulación Cerebrovascular , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Vitronectina/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
BACKGROUND AND PURPOSE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy, the most common heritable small vessel disease of the brain, is caused by dominant mutations in the NOTCH3 receptor that stereotypically lead to age-dependent Notch3ECD deposition in the vessels. NOTCH3 loss of function has been demonstrated for few mutations. However, whether this finding applies to all mutations and whether a loss-of-function mechanism drives the manifestations of the disease remain yet unknown. This study investigated the in vivo functionality of the Arg169Cys archetypal mutation. METHODS: We used mice with constitutive or conditional reduction of NOTCH3 activity, mice harboring the Arg169Cys mutation at the endogenous Notch3 locus (Notch3Arg170Cys), and mice overexpressing the Arg169Cys NOTCH3 mutant (TgPAC-Notch3R169C) on either a Notch3 wild-type or a null background. NOTCH3 activity was monitored in the brain arteries by measuring the expression of NOTCH3 target genes using real-time polymerase chain reaction. Notch3ECD deposits were assessed by immunohistochemistry. Brain parenchyma was analyzed for vacuolation and myelin debris in the white matter and infarcts. RESULTS: We identified a subset of genes appropriate to detect NOTCH3 haploinsufficiency in the adult. Expression of these genes was unaltered in Notch3Arg170Cys mice, despite marked Notch3ECD deposits. Elimination of wild-type NOTCH3 did not influence the onset and burden of white matter lesions in 20-month-old TgPAC-Notch3R169C mice, and 20-month-old Notch3-null mice exhibited neither infarct nor white matter changes. CONCLUSIONS: These data provide strong evidence that cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy can develop without impairment of NOTCH3 signaling and argue against a loss of NOTCH3 function as a general driving mechanism for white matter lesions in cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy.
Asunto(s)
CADASIL/genética , Mutación/genética , Mutación/fisiología , Receptores Notch/genética , Receptores Notch/fisiología , Animales , Encéfalo/patología , CADASIL/patología , Arterias Cerebrales/patología , Antagonistas de Estrógenos/farmacología , Regulación de la Expresión Génica , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Notch3 , Tamoxifeno/farmacología , Transcripción GenéticaRESUMEN
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.
Asunto(s)
CADASIL/diagnóstico , CADASIL/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Receptores Notch/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Anciano , Anciano de 80 o más Años , Animales , CADASIL/genética , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Femenino , Homeostasis/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Transporte de Proteínas/genética , Receptor Notch3 , Receptores Notch/genética , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
OBJECTIVE: Notch3 is critically important for the structure and myogenic response of distal arteries, particularly of cerebral arteries. However, signaling pathways acting downstream of Notch3 remain largely unknown. METHODS AND RESULTS: Transcriptome analysis using tail arteries of Notch3-null mice identified a core set of 17 novel Notch3-regulated genes confirmed in tail or brain arteries. Postnatal deletion of RBP-Jκ in smooth muscle cells recapitulated the structural, functional, and molecular defects of brain arteries induced by Notch3 deficiency. Transient in vivo blockade of the Notch pathway with γ-secretase inhibitors uncovered, in addition to Notch3, 6 immediate responders, including the voltage-gated potassium channel Kv1.5, which opposes to myogenic constriction of brain arteries, and the glutamate receptor-interacting protein 2 (Grip2) with no previously established role in the cerebrovasculature. We identified a vascular smooth muscle cell isoform of Grip2. We showed that Notch3-RBP-Jκ specifically regulates this isoform. Finally, we found that cerebral arteries of Grip2 mutant mice, which express an N-terminally truncated Grip2 protein, exhibited selective attenuation of pressure-induced contraction. CONCLUSIONS: Our data provide insight into how Notch3 signals in the brain arteries, establishing the postnatal requirement of smooth muscle RBP-Jκ in this context. Notch3-regulated transcriptome provides potential for modulating myogenic response in the cerebrovasculature.
Asunto(s)
Proteínas Portadoras/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Notch/metabolismo , Vasoconstricción , Alanina/análogos & derivados , Alanina/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Azepinas/farmacología , Proteínas Portadoras/genética , Arterias Cerebrales/metabolismo , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/crecimiento & desarrollo , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Receptor Notch3 , Receptores Notch/deficiencia , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación , Vasodilatadores/farmacologíaRESUMEN
In cerebral arteries, alterations of vascular reactivity have been observed but not well molecularly characterized. Therefore, we have hypothesized that cerebrovascular reactivity could be modified by aging via a modification of Ca(2+) signaling in smooth muscle cells. Ca(2+) signals and gene expression implicated in contraction have been measured in posterior and middle cerebral arteries from young (2-3 months) and old (20-22 months) C57Bl6/J mice. Aging induced a decrease of KCl- and caffeine-induced contraction as well as a decrease of the amplitudes and an increase of the durations of KCl- and caffeine-induced Ca(2+) signals. These results could be linked with the decrease of gene expression coding for Cav1.2, RyR2, SERCA2, PLB, STIM1, TRIC-B, and the increase of FKBP12.6 and TPCN1 gene expression. Finally, aging induced a modification of InsP3 subtype expression pattern responsible for a modification of the InsP3 affinity to activate Ca(2+) signals. These results show that aging induces a decrease of contractility correlated with modifications of the expression of genes encoding Ca(2+) signaling toolkit. Globally, the amplitude of Ca(2+) signals was decreased, whereas their duration was increased by a defection of Ca(2+) store refilling.
Asunto(s)
Envejecimiento/metabolismo , Señalización del Calcio , Arterias Cerebrales/fisiología , Envejecimiento/fisiología , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Arterias Cerebrales/citología , Arterias Cerebrales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Canales Iónicos/genética , Canales Iónicos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Contracción Muscular , Miocitos del Músculo Liso/metabolismo , Molécula de Interacción Estromal 1 , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Transcripción GenéticaRESUMEN
The Notch signalling pathway is a highly conserved cell-cell signalling mechanism that plays a central role in the development and maturation of most vertebrate organs. In vertebrates, Notch receptors, several ligands, and components of the downstream signalling machinery are expressed in the vessel. Over the past decade, numerous studies have highlighted the critical role of the Notch pathway in the vasculature. The goal of this review is to summarize our current understanding of the contribution of Notch signalling in smooth muscle cells to vascular development and physiology. We further discuss the growing clinical importance of this pathway in human pathological conditions involving the vasculature, namely cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Alagille syndrome, and pulmonary arterial hypertension.
Asunto(s)
Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Enfermedades Vasculares/fisiopatología , Animales , Humanos , Músculo Liso Vascular/crecimiento & desarrollo , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Mutación/genética , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/ultraestructura , Receptores Notch/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
OBJECTIVES: The mechanisms by which human serum albumin might protect against sepsis-induced organ dysfunction and improve survival are not elucidated. The present study was designed to assess the effects of two concentrations of human serum albumin on endotoxin-induced mortality as well as on endothelial and organ dysfunctions in both mouse and cell models. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratories. SUBJECTS: Swiss mice (n = 10-15/group) were injected with either lipopolysaccharide or vehicle. Four and 12 hrs later, mice were infused or not with human serum albumin HSA (4% or 20%, 10 mL/kg) or normal saline (0.9% NaCl, 30 mL/kg). Human uterine vein endothelial cells were exposed to both lipopolysaccharide and tumor necrosis factor-α during 8 hrs in the presence or absence of human serum albumin (4% or 20%). MEASUREMENTS AND MAIN RESULTS: Mice survival, reactivity of mesenteric arteries, and Western blot protein analysis were assessed. Circulating endothelin-1, gluthatione, gluthatione disulfide, and creatinine plasma levels were measured. Nitric oxide production, oxidative, and nitrosative stresses were also measured in situ in endothelial cells. Human serum albumin 4%, but not human serum albumin 20% or normal saline solution, improved survival time of endotoxemic mice. Furthermore, human serum albumin 4% activated endothelial nitric oxide synthase and restored lipopolysaccharide-impaired flow-dependent endothelial dilation in mesenteric arteries. This was associated with a downregulation of nuclear factor κB and an upregulation of nuclear respiratory factor-2 and heme oxygenase-1. Human serum albumin 4% reduced lipopolysaccharide-induced renal dysfunction, enhanced endothelin-1 production and glutathione plasmatic levels, whereas human serum albumin 20% increased gluthatione disulfide. Furthermore, human serum albumin 4% but not 20% blunted lipopolysaccharide-tumor necrosis factor-α-induced oxidative and nitrosative stresses in endothelial cells and increased their gluthatione levels. CONCLUSIONS: The present data confirm a protective effect of 4% human serum albumin treatment both on mice survival and endothelial dysfunction by inhibiting inflammatory and oxidative stress pathways induced by endotoxins. Conversely, higher concentrations of human serum albumin were detrimental suggesting a dose-dependent effect.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotoxemia/fisiopatología , Infecciones por Escherichia coli/fisiopatología , Albúmina Sérica/administración & dosificación , Vasodilatación/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/fisiología , Endotelio Vascular/fisiopatología , Endotoxemia/tratamiento farmacológico , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Masculino , Ratones , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/fisiopatologíaRESUMEN
Blood flow reduction induces inward remodeling of resistance arteries (RAs). This remodeling occurs in ischemic diseases, diabetes and hypertension. Nonetheless, the effect of flow reduction per se, independent of the effect of pressure or metabolic influences, is not well understood in RA. As angiotensin II is involved in the response to flow in RA, we hypothesized that angiotensin II may also be involved in the remodeling induced by a chronic flow reduction. We analyzed the effect of angiotensin I-converting enzyme inhibition (perindopril) and angiotensin II type 1 receptor blockade (candesartan) on inward remodeling induced by blood flow reduction in vivo in rat mesenteric RAs (low flow (LF) arteries). After 1 week, diameter reduction in LF arteries was associated with reduced endothelium-dependent relaxation and lower levels of eNOS expression. Superoxide production and extracellular signal-regulated kinases 1/2 (ERK1/2 phosphorylation were higher in LF than in normal flow arteries. Nevertheless, the absence of eNOS or superoxide level reduction (tempol or apocynin) did not prevent LF remodeling. Perindopril and candesartan prevented inward remodeling in LF arteries. Contractility to angiotensin II was reduced in LF vessels by perindopril, candesartan and the ERK1/2 blocker PD98059. ERK1/2 activation (ratio phospho-ERK/ERK) was higher in LF arteries, and this activation was prevented by perindopril and candesartan. ERK1/2 inhibition in vivo (U0126) prevented LF-induced diameter reduction. Thus, inward remodeling because of blood flow reduction in mesenteric RA depends on unopposed angiotensin II-induced contraction and ERK1/2 activation, independent of superoxide production. These findings might be of importance in the treatment of vascular disorders.
Asunto(s)
Angiotensina II/fisiología , Hipertensión/fisiopatología , Arterias Mesentéricas/fisiología , Resistencia Vascular/fisiología , Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Bencimidazoles/farmacología , Compuestos de Bifenilo , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Velocidad del Flujo Sanguíneo/fisiología , Enfermedad Crónica , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Perindopril/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Tetrazoles/farmacología , Resistencia Vascular/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacologíaRESUMEN
Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke in humans. Dominant mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a genetic archetype of cerebral ischemic SVD. Progress toward understanding the pathogenesis of this disease and developing effective therapies has been hampered by the lack of a good animal model. Here, we report the development of a mouse model for CADASIL via the introduction of a CADASIL-causing Notch3 point mutation into a large P1-derived artificial chromosome (PAC). In vivo expression of the mutated PAC transgene in the mouse reproduced the endogenous Notch3 expression pattern and main pathological features of CADASIL, including Notch3 extracellular domain aggregates and granular osmiophilic material (GOM) deposits in brain vessels, progressive white matter damage, and reduced cerebral blood flow. Mutant mice displayed attenuated myogenic responses and reduced caliber of brain arteries as well as impaired cerebrovascular autoregulation and functional hyperemia. Further, we identified a substantial reduction of white matter capillary density. These neuropathological changes occurred in the absence of either histologically detectable alterations in cerebral artery structure or blood-brain barrier breakdown. These studies provide in vivo evidence for cerebrovascular dysfunction and microcirculatory failure as key contributors to hypoperfusion and white matter damage in this genetic model of ischemic SVD.
Asunto(s)
Isquemia Encefálica/genética , Circulación Cerebrovascular/genética , Animales , Vasos Sanguíneos/patología , Barrera Hematoencefálica , Isquemia Encefálica/patología , CADASIL/genética , CADASIL/patología , Arterias Cerebrales/patología , Cromosomas Artificiales/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Homeostasis , Humanos , Ratones , Ratones Transgénicos , Receptor Notch3 , Receptores Notch/genéticaRESUMEN
Angiotensin II is a potent growth factor involved in arterial wall homeostasis. In resistance arteries, chronic increases in blood flow induce a rise in diameter associated with arterial wall hypertrophy. Nevertheless, the role of angiotensin II in this remodeling is unknown. We investigated the effect of blocking angiotensin II production or receptor activation on flow-induced remodeling of mesenteric resistance arteries. Arteries were ligated in vivo to generate high-flow arteries compared with normal flow (control) vessels located at a distance. Arteries were isolated after 1 week for in vitro analysis. Arterial diameter, media surface, endothelial NO synthase expression, superoxide production, and extracellular signal-regulated kinase 1/2 phosphorylation were higher in high-flow than in control arteries. Angiotensin-converting enzyme inhibition (perindopril) and angiotensin II type 1 receptor blockade (candesartan) prevented arterial wall hypertrophy without affecting diameter enlargement. The nonselective vasodilator hydralazine had no effect on remodeling. Although perindopril and candesartan increased endothelial NO synthase expression in high-flow arteries, hypertrophy remained in rats treated with N(G)-nitro-l-arginine methyl ester and mice lacking endothelial NO synthase. Perindopril and candesartan reduced oxidative stress in high-flow arteries, but superoxide scavenging did not prevent hypertrophy. Both Tempol and the absence of endothelial NO synthase prevented the rise in diameter in high-flow vessels. Extracellular signal-regulated kinase 1/2 activation in high-flow arteries was prevented by perindopril and candesartan and not by hydralazine. Extracellular signal-regulated kinase 1/2 inhibition in vivo (U0126) prevented hypertrophy in high-flow arteries. Thus, a chronic rise in blood flow in resistance arteries induces a diameter enlargement involving NO and superoxide, whereas hypertrophy was associated with extracellular signal-regulated kinase 1/2 activation by angiotensin II.
Asunto(s)
Angiotensina II/fisiología , Arterias Mesentéricas/fisiopatología , Resistencia Vascular/fisiología , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antioxidantes/farmacología , Bencimidazoles/farmacología , Compuestos de Bifenilo , Velocidad del Flujo Sanguíneo , Western Blotting , Butadienos/farmacología , Óxidos N-Cíclicos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hidralazina/farmacología , Masculino , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/patología , Ratones , Ratones Noqueados , Microscopía Confocal , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitrilos/farmacología , Perindopril/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Marcadores de Spin , Tetrazoles/farmacología , Resistencia Vascular/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Recombinant human activated protein C (rhAPC) is one of the treatment panels for improving vascular dysfunction in septic patients. In a previous study, we reported that rhAPC treatment in rat endotoxemia improved vascular reactivity, although the mechanisms involved are still under debate. In the present study, we hypothesized that rhAPC may improve arterial dysfunction through its nonanticoagulant properties. Ten hours after injection of LPS in mice (50 mg/kg ip), aortic rings and mesenteric arteries were isolated and incubated with or without rhAPC for 12 h. Aortic rings were mounted in a myograph, after which arterial contractility and endothelium-dependent relaxation were measured in the presence or absence of nitric oxide synthase or cyclooxygenase inhibitors. Flow (shear stress)-mediated dilation with or without the above inhibitors was also measured in mesenteric resistance arteries. Protein expression was assessed by Western blotting. Lipopolysaccharide (LPS) reduced aortic contractility to KCl and phenylephrine as well as dilation to acetylcholine. LPS also reduced flow-mediated dilation in mesenteric arteries. In rhAPC-treated aorta and mesenteric arteries, contractility and endothelial responsiveness to vasodilator drug and shear stress were improved. rhAPC treatment also improved LPS-induced endothelial dysfunction; this effect was associated with an increase in the phosphorylated form of endothelial nitric oxide synthase and protein kinase B as well as cyclooxygenase vasodilatory pathways, thus suggesting that these pathways, together with the decrease in nuclear factor-kappaB activation and inducible nitric oxide synthase expression in the vascular wall, are implicated in the endothelial effect of rhAPC. In conclusion, ex vivo application of rhAPC improves arterial contractility and endothelial dysfunction resulting from endotoxemia in mice. This finding provides important insights into the mechanism underlying rhAPC-induced improvements on arterial dysfunction during septic shock.
Asunto(s)
Arterias/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotoxemia/tratamiento farmacológico , Proteína C/farmacología , Acetilcolina/antagonistas & inhibidores , Acetilcolina/farmacología , Animales , Aorta/efectos de los fármacos , Western Blotting , Inhibidores de la Ciclooxigenasa/farmacología , Endotoxemia/inducido químicamente , Endotoxemia/patología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Lipopolisacáridos , Masculino , Arterias Mesentéricas/fisiología , Ratones , FN-kappa B/biosíntesis , FN-kappa B/genética , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteína C/uso terapéutico , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Resistencia Vascular/fisiología , Vasodilatadores/antagonistas & inhibidores , Vasodilatadores/farmacologíaRESUMEN
OBJECTIVE: Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation and might be potentially involved in the pathophysiology of septic shock. This study was designed to assess the effects of MPs from septic origin on the systemic hemodynamics as well as on the inflammatory, oxidative, and nitrosative stresses. DESIGN: A prospective, randomized, controlled experimental study with repeated measurements. SETTING: Investigational animal laboratory. SUBJECTS: Forty healthy rats were randomly allocated to three groups: 10 animals inoculated with MPs isolated from control rats (cMPs), 15 animals inoculated with MPs isolated from sham rats (shMPs), and 15 animals inoculated with MPs isolated from rats with peritonitis (sMPs). INTERVENTIONS: Rats were anesthetized, mechanically ventilated, and infused with the same amount of cMPs, shMPs, or sMPs. We measured the heart rate, mean arterial pressure, carotid artery, and portal vein blood flows. Hemodynamic parameters were recorded during 7 hours, and then animals were killed. Aorta and heart were harvested for further in vitro tissue analyses. MEASUREMENTS AND MAIN RESULTS: 1) The cellular origin (phenotype) but not the circulating concentration of MPs was different in septic rats, characterized by a significant increase in leukocyte-derived MPs. 2) sMPs but not cMPs or shMPs decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. 3) Rats inoculated with sMPs exhibited an increase in superoxide ion production and nuclear factor kappa B activity, overexpression of inducible nitric oxide synthase with subsequent nitric oxide overproduction and decrease in endothelial nitric oxide synthase activation. CONCLUSIONS: Rats with sepsis induced by peritonitis exhibited a specific phenotype of MPs. Inoculation of sMPs in healthy rats reproduced hemodynamic, septic inflammatory patterns, associated with oxidative and nitrosative stresses.