Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease

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.     

A novel mutation (C271F) in the Notch3 gene in a Chinese man with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset hereditary condition caused by mutations in the Notch3 gene. A Chinese man was studied. METHOD: Electronic microscopy examination of skin biopsy. The Notch3 gene was screened for mutations by polymerase chain reaction and direct DNA sequencing. RESULTS: Electronic microscopy showed the presence of deposits of granular osmiophilic material in dermal capillaries in the index patient. A novel heterozygous C271F in exon 6 was detected in the index patient. This heterozygous C271F mutation was also detected in the asymptomatic elder son but was not detected in the asymptomatic wife of the patient. Allele specific amplification showed that C271F was not detected in 100 normal subjects. CONCLUSION: We established the molecular basis of CADASIL in a Chinese man. Mutation detection assay provides a reliable method for confirming the diagnosis of CADASIL.     

GENETICS

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebrovascular disease leading to accumulating neurologic deficits and dementia. CADASIL has been linked to nucleotide substitutions and deletions in the Notch3 gene. All the mutations described until now lead to unpaired cysteine residue in the epidermal growth factor-like repeats. The authors report a family with CADASIL carrying a deletion in the Notch3 gene that did not involve a cysteine residue.
Comment: At least 400 families from countries around the world have been described with CADASIL. The four classic features are migraine with aura, stroke, dementia, and psychiatric disturbances. Migraine with aura, especially familial hemiplegic migraine and migraine with prolonged aura, is linked genetically to CADASIL on chromosome 19, occurs in up to 30% of patients, and when present is usually the earliest symptom. Definitive diagnosis is by skin biopsy, with characteristic depositions surrounding degenerating smooth muscle cells in the media. —Stewart J. Tepper, MD     

Recognizing CADASIL: a Secondary Cause of Migraine with Aura

Purpose of Review

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an important disease to consider in the differential diagnosis of migraine with aura. This review examines current literature regarding migraine in CADASIL, as well as diagnostic and treatment modalities.

Recent Findings

Recent studies have shown that smoking is a modifiable risk factor for progression of CADASIL (Chabriat et al. in Stroke 47:4–11, 2015). Specific imaging changes and findings on clinical exam can predict disease progression. However, migraine symptoms often precede MRI changes (Guey et al. in Cephalalgia 36:1038–47, 2015). A recent paper on migraine treatment in CADASIL reevaluates the prevailing belief that vasoconstrictive abortive medications are contraindicated in these patients (Tan and Markus in PLoS ONE 11:e0157613, 2016).

Summary

CADASIL is an autosomal dominantly inherited vasculopathy causing ischemic pathology in younger individuals due to a mutation in the NOTCH3 gene. The mutation results in impaired arterial contractility due to accumulation of granular osmiophilic extracellular material (GOM) in vascular smooth muscle cells (VSMCs). Clinical manifestations include migraine with and without aura, cognitive decline, ischemic events, and mood disorders. The presenting symptom is often migraine with aura. Characteristic MRI changes are often present. Genetic screening is available to confirm NOTCH3 mutation and pathognomic changes are often seen in skin biopsy. Treatment of migraine is similar to the general population, but with some notable and specific differences. Further studies in CADASIL, other small vessel arteriopathies, and migraine may help us understand more about the pathophysiology of these conditions and help with treatment development.

     

变性高压液相色谱分析在中国家族性CADASIL患者基因筛查中的应用

目的:探讨变性高压液相色谱分析(DHPLC)在伴皮质下梗死及白质脑病的常染色体显性遗传性脑动脉病(CADASIL)患者诊断中的方法和价值。方法:运用DHPLC及DNA直接测序技术,对CADASIL家系先证者、家系成员14例(CADASIL组)及健康对照者100例(对照组)进行Notch3基因检测。结果:CADASIL组发现3种致病性突变,其中Cys134Tyr为新的突变类型,同时发现15种多态类型;对照组中未发现突变。结论:DHPLC技术在筛查Notch3中起重要作用,但在筛查时应进行多个柱温的测试,温度调换以2℃为宜。     

Investigating the association between Notch3 polymorphism and migraine

OBJECTIVE: The aim of the present study was to evaluate whether the functional Notch3 polymorphism T6746C, which is not causative for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), might be a risk factor for migraine. BACKGROUND: It has been recently demonstrated that migraine is characterized by subclinical brain infarctions and white matter lesions. Several genetic risk factors have been associated with migraine, but no study has unraveled a possible relationship between migraine and Notch3, which is involved in vascular damage. Mutations in Notch3 gene have been demonstrated to be pathogenetic for CADASIL, a small vessel disease of the brain characterized by migraine. METHODS: A total of 156 migraine patients and 128 nonheadache healthy volunteers entered the study. Demographic and clinical characteristics were carefully recorded, and a neurological work-up was performed. Moreover, each subject underwent a blood sampling for Notch3 genotype determination. RESULTS: Notch3 genotypes as well as allele frequencies did not differ in migraine patients compared to controls, even adjusting for the presence of possible confounds. No difference has been found either in migraine patients with aura or in those without aura. CONCLUSIONS: These findings support the view that functional polymorphism T6746C in Notch3 gene is not involved in increasing the risk of migraine or migraine subtypes.     

CADASIL病

CADASIL(伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病 )的研究是近年来证实遗传因素参与脑血管病发病的重要进展之一 ,其主要临床特点为家族遗传方式起病、中年发病、逐渐进展的缺血性脑卒中样病程、假性球麻痹、进行性血管性痴呆、先兆症状的偏头痛发作和精神症状。脑内广泛多发的白质灶、明确的MRI白质异常信号以及病理学明确的小动脉病变是本病的基本特征。分子遗传学研究发现 19号染色体Notch3基因突变与本病有关     

Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions.

Human atheromata obtained in vivo were used to test the hypothesis that transforming growth factor-beta 1 plays a role in the development of vascular restenosis. We analyzed 28 specimens from patients with primary atherosclerotic or restenotic lesions; 26 of these were obtained by directional atherectomy and 2 at the time of coronary bypass surgery. Seven control tissues included operatively excised segments of human internal mammary artery, myocardium, and unused portions of vein graft obtained intraoperatively. From these 35 specimens, 210 sections were examined using in situ hybridization. Measurement of silver grains/nucleus disclosed that expression of transforming growth factor-beta 1 mRNA was highest in restenotic tissues (P < 0.001 vs. primary atherosclerotic tissues) and lowest in nonatherosclerotic (control) tissues. In cultures of human vascular smooth muscle cells grown from explants of internal mammary artery, expression of mRNA for transforming growth factor-beta 1 was significantly greater in subconfluent than in confluent smooth muscle cells (P = 0.05). Transforming growth factor type-beta III receptor was expressed in cell cultures and undetectable in the tissue specimens. Sections taken adjacent to those studied by in situ hybridization were examined by immunohistochemistry using antibodies against transforming growth factor-beta 1 and alpha-actin (as a marker for smooth muscle cells) and disclosed transforming growth factor-beta 1 in smooth muscle cells present in these sections. These findings are consistent with the concept that transforming growth factor-beta 1 plays an important role in modulating repair of vascular injury, including restenosis, after balloon angioplasty.     

CADASIL and migraine: A narrative review

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene and is clinically characterized by recurrent stroke, cognitive decline, psychiatric disturbances and migraine. The prevalence of migraine in CADASIL is slightly higher than in the general population, and the proportion of migraine with aura is much higher. The pathophysiological mechanism that leads to increased aura prevalence in CADASIL is unknown. Possible mechanisms of the excess of migraine with aura are an increased susceptibility to cortical spreading depression (CSD) or a different expression of CSD. It is also possible that the brainstem migraine area is involved in CADASIL. Last, it is possible that the NOTCH3 mutation acts as a migraine aura susceptibility gene by itself. In this narrative review we summarize the literature about migraine in CADASIL, with a special focus on what CADASIL might teach us about the pathophysiology of migraine.     

有机阴离子转运蛋白1在人血管平滑肌细胞表达的意义

[目的]检测有机阴离子转运蛋白1(OAT1) mRNA和蛋白质在人血管平滑肌细胞的表达以及尿酸刺激对其表达的影响.[方法]分离培养人脐动脉血管平滑肌细胞,提取细胞总RNA,RT-PCR方法扩增特异性OAT1 cDNA片断,制备探针,用Northern blot方法检测OAT1 mRNA的表达,提取细胞膜蛋白和总蛋白,用Western blot方法检测OAT1的蛋白表达;用800 μmol/L的尿酸刺激细胞,观察OAT1的表达变化.[结果]从人血管平滑肌细胞检测到OAT1 mRNA和蛋白的表达.尿酸可以明显增强OAT1的表达.[结论]人血管平滑肌细胞有明确的OAT1表达,OAT1可能部分介导了血管平滑肌细胞对尿酸的摄取过程.     

Evidence for altered Na+/H+ antiport activity in cultured skeletal muscle cells and vascular smooth muscle cells from the spontaneously hypertensive rat.

1. Intracellular pH and Na+/H+ antiport activity were determined by a fluorimetric method in cultured skeletal muscle cells (myoblasts) and aortic vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats. 2. The intracellular pH was significantly more alkaline at three different extracellular pH values in both myoblasts and vascular smooth muscle cells from the spontaneously hypertensive rats than in those from the normotensive control rats. 3. A kinetic analysis of the Na+/H+ antiport activity in these cells showed that the raised activity in the spontaneously hypertensive rats was due to an increased maximal transport capacity in vascular smooth muscle cells and to an increase in the affinity of the antiport for internal H+ in the myoblasts. 4. When the extracellular pH was reduced in the skeletal muscle cells of both types of rat, the intracellular pH fell. However, in vascular smooth muscle cells, a reduction in the extracellular pH was not associated with a fall in the intracellular pH. This resistance of the intracellular pH to changes in the extracellular pH differentiates vascular smooth muscle cells from other cells that have been studied in this way.     

Genetic variants of the NOTCH3 gene in migraine--a mutation analysis and association study

Mutations in the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Exons 3 and 4 are mutation hotspots. Migraine is a clinical hallmark of CADASIL. The objective of this study was to investigate whether genetic variants in exons 3 and 4 of the NOTCH3 gene are associated with migraine. Exons 3 and 4 of the NOTCH3 were analysed for mutations and polymorphisms by direct DNA sequencing in 97 migraineurs and the same number of control individuals. No mutations in exons 3 and 4 of the NOTCH3 gene were found in 97 patients with migraine. However, association analysis revealed significant association of the single nucleotide polymorphism (SNP) rs1043994 with migraine.     

Acute Confusional Migraine May Be a Presenting Feature of CADASIL

Objective.— Characterize the phenomenon of acute confusional migraine (ACM) among Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) patients and emphasize the possibility of CADASIL in adults with ACM.
Background.— ACM, well described in children, has rarely been reported in adults. Although 30-60% of CADASIL patients have migraine, acute confusional state during migraine has not been described. We describe 7 patients with ACM that complicated up to 50% of the migraine episodes.
Design/Methods.— Detailed neurologic evaluation was performed in 20 CADASIL patients; International Classification of Headache Disorders 2nd edition criteria were used to diagnose migraine.
Results.— The mean age was 51 years. Fourteen patients reported headache and 11 met the criteria for migraine (mean age of onset 25). Seven patients experienced concomitant confusion, within 3 years of migraine onset. Confusion occurred either abruptly or insidiously, at the onset of aura or headache, lasting for 2-48 hours, and ending abruptly. These episodes were stereotypic, characterized by disorientation with agitation, and retrograde amnesia for the episodes. Patients reported disorientation to time and place, inability to recognize friends and relatives, difficulty with finding directions home, fear of getting lost, inability to analyze traffic lights or tell time. Patients reliably predicted the episodes and felt the need to seek a safe place for protection. Severity of the episodes progressed, but a striking improvement occurred after the first stroke.
Conclusion.— ACM may be a presenting feature and important clue, enabling CADASIL to be recognized up to a decade or earlier than at present. Therefore, a brain MRI and/or testing for Notch3 mutations should be considered in adult patients with ACM.     

Two cases of Japanese CADASIL with corpus callosum lesion

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a rare hereditary stroke disease. In the present study, a Japanese CADASIL family was first reported with missense mutation of Arg141Cys of Notch3 and a unique lesion of corpus callosum. Upon neuropsychological examination, our case 1 showed only right-handed constructional apraxia associated with corpus callosum lesion. No other callosal disconnection signs were present. Sagittal T2 weighted image of case 1 showed multiple small lesions along with the pericallosal branches from the truncus to the posterior part of the splenium in the corpus callosum. Although detailed mapping of the corpus callosum for functional fractionation in humans remains incomplete, the constructional apraxia on the right may be related to callosal dysfunction from the truncus to the posterior part of the splenium in the corpus callosum.     

CADASIL: Imaging Characteristics and Clinical Correlation

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene located on chromosome 19p13. CADASIL causes a clinical syndrome of migraines (frequently with aura), progressive strokes, and cognitive decline in adults leading to severe functional impairment by the seventh decade of life. Genetic testing is the gold standard for diagnosing this condition, but the syndrome can be suspected clinically based on history and a characteristic pattern of confluent subcortical white matter disease in the anterior temporal poles and external capsule. Additional abnormalities include cerebral microbleeds and large vessel stenosis, particularly in Asian populations. Familiarity with radiologic findings in CADASIL is essential to the correct diagnosis and subsequent management of this disease.     

An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation

The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.     

细胞在小口径组织工程血管中的抗钙化作用

背景：小口径组织工程血管的远期结果研究极少,尚未见到研究组织工程血管分子水平、离子水平远期结果和平滑肌细胞与钙化关系的报道。目的：利用脱细胞猪股动脉基质作为支架和犬血管壁细胞作为种子细胞体外构建小口径组织工程血管,植入种子细胞供体犬股动脉部位6个月,观察植入物中层平滑肌细胞与钙化的关系。方法：12只实验犬被随机分为支架组（n=6）和再细胞化组（n=6）,自体股动脉被作为对照组;支架组犬接受猪股动脉经脱细胞后的基质支架植入双侧股动脉,再细胞化组犬接受受体血管壁细胞共同培养、联合种植于脱细胞的猪股动脉基质并体外预适应后植入血管壁细胞供体双侧股动脉位置;6个月后测定植入物和对照组股动脉组织钙含量、平滑肌密度和病理学变化。结果与结论：小口径组织工程血管植入后6个月检查见2组植入物无明显狭窄和扩张,扫描电镜示内表面均已完全内皮化,有管壁僵硬和局部钙化斑块形成,以上改变以支架组植入物更明显。支架组管道组织钙含量显著高于再细胞化组和自体股动脉（P〈0.01）,再细胞化组植入物组织钙含量亦显著高于自体股动脉（P〈0.01）;病理学检查示再细胞化组植入物平滑肌密度高于支架组（P〈0.01）,再细胞化组和支架组植入物平滑肌密度均低于对照组（P〈0.01）;超声检查见2组管道植入术后即刻与6个月后舒缩幅度较邻近自体股动脉舒缩幅度小,有部分管道无舒缩功能。结果提示,猪股动脉常规脱细胞方去获得的基质作为支架体外构建组织工程血管时,平滑肌细胞难于迁移至支架中层,中层平滑肌密度低,植入体内6个月后中层平滑肌细胞密度仍低,平滑肌细胞有抗血管钙化作用。     

人类巨细胞病毒感染对血管平滑肌细胞脂代谢相关基因表达的影响

目的：探讨血管平滑肌细胞被感染人类巨细胞病毒后,其细胞内胆固醇脂代谢及相关基因表达的变化情况。方法通过分离原代脐动脉血管平滑肌细胞,以1个感染复数的人类巨细胞病毒攻击作为实验组,对照组则采用含3％小牛血清的DMEM/F12培养基模拟感染。检测平滑肌细胞内胆固醇含量在病毒攻击后的变化情况,以基因表达谱芯片技术检测基因表达情况。结果血管平滑肌细胞被感染人类巨细胞病毒后,细胞内相关基因表达发生显著变化,其中低密度脂蛋白受体相关蛋白10、11、12以及 HMG-CoA 合成酶,HMG-CoA 还原酶,B 型清道夫受体表达上调。载脂蛋白 A1、载脂蛋白 M 表达下调。结论人类巨细胞病毒感染血管平滑肌细胞后可能通过改变脂代谢途径的相关基因表达,增强细胞内胆固醇合成,导致胆固醇代谢失衡,从而参与动脉粥样硬化的发病过程。     

A case of CADASIL caused by NOTCH3 c.512_605delinsA heterozygous mutation

BackgroundAutosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebrovascular disease closely related to the NOTCH3 gene. More than 200 mutations in this gene have been reported to be associated with this disease.MethodsThe NOTCH3 gene from CADASIL patient was screened for mutations by whole‐exome sequencing (WES). PCR amplification and direct Sanger sequencing were used to verify the suspicious gene mutation sites detected by WES.ResultsWe performed second‐generation sequencing on a sample of the patient''s genome and found a heterozygous deletion‐insertion mutation c.512_605delinsA in exon 4 of NOTCH3, which resulted in amino acid changes p.G171_A202delinsE. This variation was confirmed by the direct Sanger sequencing. It may be rated as a CADASIL clinical variation.ConclusionDiscovery of this mutation site provides an important theoretical basis for specific gene‐based diagnosis and treatment of CADASIL.