血管结构蛋白、生成因子和基质蛋白酶在颅内动脉瘤破裂进程中的表达规律研究

目的 拟通过同一患者的不同阶段的颅内动脉瘤(IA)血管结构蛋白、生长因子和基质蛋白酶的表达进行分析,探索动脉瘤破裂的分子机制,为IA的早期筛查和预防提供理论依据。方法 采集6名IA患者未破裂动脉瘤、破裂动脉瘤的动脉瘤壁和颞浅动脉血管壁样本。通过qRT-PCR和Western blotting检测3种结构蛋白(III型胶原、纤维连接蛋白和层粘连蛋白)、2种血管生成因子(血管内皮生长因子、碱性成纤维细胞生长因子）和2种基质蛋白酶的表达水平变化趋势。结果 正常血管壁样本（颞浅动脉）中III型胶原、层粘连蛋白、成纤维细胞生长因子和基质金属蛋白酶2呈现高水平表达;未破裂动脉瘤样本中,大多数蛋白趋于中度表达水平;动脉瘤破裂后III型胶原和层粘连蛋白表达水平降低,纤维连接蛋白和血管内皮生长因子表达水平升高。mRNA水平表达与蛋白表达水平基本一致。结论 正常血管壁、未破裂动脉瘤壁和破裂动脉瘤壁中结构蛋白和血管生成因子表现出不同的表达水平和模式。 [国际神经病学神经外科学杂志, 2022, 49(3): 14-20.]     

Role of endoglin and transforming growth factor‐beta in progressive white matter damage after an ischemic stroke

We morphologically examined human brains several years after a territorial ischemic stroke to assess the development of progressing white matter damage and its pathomechanisms. Our investigations focused on the role of TGF‐β, one of the factors whose expression increases after tissue damage, and its receptor endoglin in the propagation of postischemic injury. Examination of the white matter adjacent to the postapoplectic cavity revealed structural changes in the capillary vessels, disturbed microcirculation, and deep endothelial cell damage with DNA fragmentation in the TUNEL reaction. Many oligodendrocytes also revealed DNA damage and an increased expression of caspase‐3. In the rarefied white matter, the microvessel immune reaction to TGF‐β was diminished while the expression of endoglin was heterogeneous: absent in some capillaries but increased in others in comparison to the vessels located more peripherally from the cavity and in the control material. We conclude that endoglin and TGF‐β can be involved in the development of the microangiopathy responsible for the propagation of postischemic white matter injury in humans. We suggest that disturbances in endoglin expression can influence TGF‐β signaling and, consequently, vessel structure and function. Pronounced endoglin expression can lead to decreased vessel wall integrity while a lack of the constitutively expressed protein is probably a mirror of deep vessel damage.     

脑动静脉畸形破裂出血病人中Ang1、Ang2、VEGF、LP的表达和意义

目的研究血管生成素1(angiopoietin 1,Ang1)、血管生成素2(angiopoietin 2,Ang2)、血管内皮生长因子(vascular-endothelial growth factor,VEGF)及瘦素(leptin,LP)在脑动静脉畸形(AVM)破裂出血病人中的表达和意义。方法收集手术切除的AVM标本28例,将14例有近期破裂出血史的病人作为AVM出血组,14例无明显破裂出血史的病人作为AVM未出血组,采用免疫组织化学法检测2组标本Ang1、Ang2、VEGF及LP的表达,并进行统计分析。结果免疫组织化学法显示:Ang1、Ang2、VEGF及LP在2组血管内皮细胞中均有阳性表达;与AVM未出血组比较,AVM出血组的血管内皮细胞中Ang2、VEGF、LP表达显著增强(均P<0.01),而Ang1的表达较弱(P>0.05)。结论 Angl、Ang2、VEGF及LP在AVM的畸形血管中存在特异性表达,尤其是Ang2、VEGF及LP可能与AVM的进展及破裂出血有密切关系。     

Interleukin‐18 and transforming growth factor‐beta 1 plasma levels in Alzheimer’s disease and vascular dementia

Inflammation has been involved in the development of dementia in cerebrovascular diseases. To investigate the cellular activation of the peripheral immune system in patients with Alzheimer’s disease (AD) and vascular dementia (VaD), we determined the presence of IL‐18 and TGF‐β1 in the plasma by using ELISA. The levels of IL‐18 and TGF‐β1 were significantly elevated in patients with AD and VaD compared to non‐demented, age‐matched subjects. We found an inverse correlation between the levels of IL‐18 and TGF‐β1 in AD patients. In VaD patients, the correlation between IL‐18 and TGF‐β1 reached a borderline positive value. Whereas, in the non‐demented, age‐matched subjects, a positive correlation between IL‐18 and TGF‐β1 levels was observed. These findings indicate that IL‐18 and TGF‐β1 elevation is associated with AD and VaD patients, confirming that the immune system might exert a remarkable role in the development and progression of neurodegenerative disorders. Moreover, as different modifications were detected in the patients affected by AD and VaD, we propose that IL‐18 and TGF‐ β1 plasma levels might represent possible differential biomarkers.     

Differential effects of transforming growth factor-β1 on interleukin-1-induced cellular inflammation and vascular permeability in the rabbit retina

Intra-vitreal injection of 300 U of interleukin (IL)-1β into the rabbit eye induces an inflammation of the retina characterized by hemorrhage, monocyte and neutrophil infiltration, and an increase in vascular permeability that peaks 24 h post-injection. Since the epiretinal vessels involved in this inflammation form part of the blood-retina barrier, we used this model to investigate the effects of the immunosuppressive cytokine TGFβ1 on inflammation within the context of the central nervous system. We found that intra-vitreal injection of 1 μg rh TGFβ administered concomitantly with rh IL-1β significantly reduced IL-1β-induced hemorrhage by 78%, and monocyte and neutrophil infiltration by 53% and 62%, respectively. In contrast, TGFβ did not reduce the IL-1β-induced increase in vascular permeability. However, TGFβ by itself caused a statistically significant increase in serum proteins in perfused tissues of the eye, to give a 3.1±0.4 fold increase in protein content over control values. No cellular inflammation accompanied this alteration in vascular permeability. These data indicate that whereas the local administration of TGFβ may be an effective inhibitor of cellular inflammation in the CNS, the effects on alterations in vascular permeability and accumulation of serum proteins may be more complex.     

金属蛋白酶2、血管内皮生长因子和肿瘤抑制因子在人脑胶质瘤中的表达和临床意义

目的:探讨人脑胶质瘤中的金属蛋白酶2(MMP-2)、血管内皮生长因子(VEGF)和肿瘤抑制因子(P16)的表达。方法:应用免疫组化法检测55例胶质瘤组织中的MMP-2、VEGF和P16蛋白的表达,通过原位杂交检测MMP-2 mRNA的表达。结果:胶质瘤的恶性程度与MMP-2、VEGF表达和P16的缺失表达呈正相关,随着胶质瘤的恶性程度的增高而增加。结论:胶质瘤的恶性程度由多种因素决定。胶质瘤中MMP-2含量越高,肿瘤细胞在浸润的过程中突破血脑屏障的能力越强;VEGF的表达增加,肿瘤的血液供应越丰富,恶性程度越高。P16蛋白缺失率高,组织学分化程度低,在一定程度上反映了胶质瘤细胞的恶性生物学行为。     

MAPK/ERK激酶-ERK信号通路参与外源性bFGF对缺氧-复氧损伤后星形胶质细胞内Egr-1结合活性的调节(英文)

目的静脉注射碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)可以明显降低实验性脑缺血大鼠的脑梗死面积,但该作用的分子机制尚不清楚。本文旨在研究外源性bFGF 作用的信号转导通路。方法缺氧-复氧损伤星形胶质细胞。Western blot检测外源性bFGF作用后丝裂原活化蛋白激酶/细胞外信号调节激酶激酶(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase,MEK)-细胞外信号调节激酶(extracellular signal-regulated kinase, ERK) 信号通路活化。电泳变动迁移率分析实验检测外源性bFGF 作用后核转录因子早期生长反应因子-1(early growth respons factor 1, Egr-1)的结合活性变化。结果外源性bFGF可以保护胞外信号调节激酶MEK-ERK信号通路蛋白不被氧自由基降解。MEK-ERK信号通路在外源性bFGF作用后活化。这一信号通路进一步使Egr-1结合活性升高。结论外源性bFGF可能通过激活ERK信号通路,促进内源性转录因子Egr-1的结合活性升高,进而促进内源性bFGF的表达。     

MAPK/ERK激酶-ERK信号通路参与外源性bFGF对缺氧-复氧损伤后星形胶质细胞内Egr-1结合活性的调节

目的 静脉注射碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）可以明显降低实验性脑缺血大鼠的脑梗死面积,但该作用的分子机制尚不清楚。本文旨在研究外源性bFGF 作用的信号转导通路。方法缺氧-复氧损伤星形胶质细胞。Western blot检测外源性bFGF作用后丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（mitogen-activated protein kinase/extracellular signal-regulated kinase kinase,MEK）-细胞外信号调节激酶（extracellular signal-regulated kinase, ERK） 信号通路活化。电泳变动迁移率分析实验检测外源性bFGF 作用后核转录因子早期生长反应因子-1（early growth respons factor 1, Egr-1）的结合活性变化。结果外源性bFGF可以保护胞外信号调节激酶MEK-ERK信号通路蛋白不被氧自由基降解。MEK-ERK信号通路在外源性bFGF作用后活化。这一信号通路进一步使Egr-1结合活性升高。结论外源性bFGF可能通过激活ERK信号通路,促进内源性转录因子Egr-1的结合活性升高,进而促进内源性bFGF的表达。     

Alpha-smooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factor-beta 1 (TGF-beta1)

Aims: Rapid and extensive activation of astrocytes occurs subsequent to many forms of central nervous system (CNS) injury. Recent studies have revealed that the expression profile of reactive astrocytes comprises antigens present during astrocyte development. Elevated levels of the injury‐related cytokine transforming growth factor‐beta 1 (TGF‐β1) secreted by microglial cells and invading macrophages have been correlated with the reactive astrocyte phenotype and glial scar formation. Methods: In the present study, the expression profile of alpha‐smooth muscle actin (α‐SMA) and nestin, two cytoskeletal proteins expressed during astrocyte development, was studied in multiple sclerosis (MS) lesions. In addition, α‐SMA and nestin organization and expression were analysed in rat primary astrocyte cultures in response to TGF‐β1. Results: In active lesions and in the hypercellular margin of chronic active MS lesions, immunostaining for α‐SMA revealed a subpopulation of reactive astrocytes, whereas the majority of reactive astrocytes expressed nestin. α‐SMA and nestin expressing reactive astrocytes were in close relationship with TGF‐β1 expressing macrophages or microglia. In addition, TGF‐β1 expression within α‐SMA or nestin expressing astrocytes was also detected. Our in vitro experiments showed that TGF‐β1 regulated the organization and expression of α‐SMA and nestin in astrocytes. Conclusions: Reactive astrocytes in active MS lesions re‐express α‐SMA and nestin. We suggest that the in vivo re‐expression might be under regulation of TGF‐β1. These results further clarify the regulation of astrocyte activity after CNS injury, which is important for the astroglial adaptation to pathological situations.     

Novel role of TGF-beta in differential astrocyte-TIMP-1 regulation: implications for HIV-1-dementia and neuroinflammation

Astrocyte production of tissue inhibitor of metalloproteinase (TIMP)-1 is important in central nervous system (CNS) homeostasis and inflammatory diseases such as HIV-1-associated dementia (HAD). TIMPs and matrix metalloproteinases (MMPs) regulate the remodeling of the extracellular matrix. An imbalance between TIMPs and MMPs is associated with many pathologic conditions. Our recently published studies uniquely demonstrate that HAD patients have reduced levels of TIMP-1 in the brain. Astrocyte-TIMP-1 expression is differentially regulated in acute and chronic inflammatory conditions. In this and the adjoining report (Gardner et al., 2006), we investigate the mechanisms that may be involved in differential TIMP-1 regulation. One mechanism for TIMP-1 downregulation is the production of anti-inflammatory molecules, which can activate signaling pathways during chronic inflammation. We investigated the contribution of transforming growth factor (TGF)-signaling in astrocyte-MMP/TIMP-1-astrocyte regulation. TGF-beta1 and beta2 levels were upregulated in HAD brain tissues. Co-stimulation of astrocytes with IL-1beta and TGF-beta mimicked the TIMP-1 downregulation observed with IL-1beta chronic activation. Measurement of astrocyte-MMP protein levels showed that TGF-beta combined with IL-1beta increased MMP-2 and decreased proMMP-1 expression compared to IL-1beta alone. We propose that one of the mechanisms involved in TIMP-1 downregulation may be through TGF-signaling in chronic immune activation. These studies show a novel extracellular regulatory loop in astrocyte-TIMP-1 regulation.     

Role of VEGF and matrix metalloproteinase‐9 in peritumoral brain edema associated with supratentorial benign meningiomas

Accumulating evidence indicates that VEGF and matrix metalloproteinase‐9 (MMP‐9) play a central role in the development of peritumoral brain edema (PTBE) associated with human brain tumors. However, the roles of these proteins, particularly of MMP‐9, in PTBE associated with benign meningiomas have not been elucidated. We investigated the association between clinical features and biological factors, such as VEGF and MMP‐9, and the incidence of PTBE and edema index (EI) in 60 patients with benign meningiomas. In this study, supratentorial lesions were examined for evaluating the extent of PTBE in the surrounding normal brain tissue. VEGF and MMP‐9 expression was immunohistochemically examined. Multivariate analysis revealed that the presence of pial blood supply (odds ratio [OR] 12.250; P = 0.0096) and VEGF (OR 7.683; P = 0.0155), but not MMP‐9 (OR 1.178; P = 0.8113), expression are significant factors that independently predict the incidence of PTBE and influence EI. VEGF (P = 0.0397) and MMP‐9 (P = 0.0057) expression correlates with the presence of pial blood supply. Moreover, tumors with high VEGF and MMP‐9 expression had higher EIs than those with high expression of either (P = 0.030). Our findings suggest that MMP‐9 expression was positively related to VEGF expression and pial blood supply and promoted the occurrence of PTBE by inducing the disruption of the arachnoid membrane and formation of pial blood supply.     

Plasminogen activator inhibitor-1 is an aggregate response factor with pleiotropic effects on cell signaling in vascular disease and the tumor microenvironment

In hemostasis, the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) functions to stabilize clots via inhibition of tissue plasminogen activator (tPA) with subsequent inhibition of fibrinolysis. In tissues, PAI-1 functions to inhibit extracellular matrix degradation via inhibition of urokinase plasminogen activator (uPA). Elevated levels of PAI-1 in the vasculature and in tissues have long been known to be associated with thrombosis and fibrosis, respectively. However, there is emerging evidence that PAI-1 may participate in the pathophysiology of a number of diseases such as atherosclerosis, restenosis, and cancer. In many of these disease states, the canonical view of PAI-1 as an inhibitor of tPA and uPA cannot fully account for a mechanism whereby PAI-1 contributes to the disease. In these cases, one must consider recent data, which indicates PAI-1 can directly promote pro-proliferative and anti-apoptotic signaling in a variety of cell types. Given the wide variety of inflammatory, hormonal, and metabolic signals that increase PAI-1 expression, it is important to consider mechanisms by which PAI-1 can directly participate in disease etiology.     

In vitro activation of extracellular signal-regulated kinase1/2 in the inner ear of guinea pigs

Growth factors such as vascular endothelial growth factor (VEGF) exert their proliferative properties partly through activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). Although both VEGF and inactive ERK could be detected in the inner ear of guinea pigs, under normal conditions activated ERK (phospho-ERK) was found only sparely. Cochleae of adult guinea pigs were removed, incubated with VEGF in a carbogen-gased organ-bath for 5, 15, 30 and 60 min (n=6 in each group), fixed with PFA 4%, embedded in paraffin and sectioned, followed by immunohistochemical staining to inactive and active ERK. Whereas inactive ERK was found in all cochleae, in sensory and supporting cells of the apex activated ERK was strongly detected after 5-min VEGF-incubation. After 15 min all Corti-organs showed clear staining corresponding to activated ERK, which decreased again after 30 min. Faint staining in endothelial cells of the spring-coil-vessels and in the spiral ganglion cells was found after 30 min and was increased after 60 min, while the staining in the Corti-organs vanished. Addition of the MEK-inhibitor PD 98059 to the organ-bath led to diminished phospho-ERK1/2 immunostaining. These findings provide evidence for a VEGF-dependent phosphorylation of ERK1/2 in the cochlea. Activated ERK1/2 is thought to support axonal outgrowth, enhancement of cell survival and to regulate the turnover of the NO/cGMP-pathway.     

脑缺血耐受大鼠脑组织血管内皮生长因子和血管生成素-1表达的改变及其意义

目的探讨血管内皮生长因子(VEGF)和血管生成素-1(Ang-1)在短暂性脑缺血预处理(IP)诱导的脑缺血耐受大鼠脑组织表达的改变及其意义。方法将99只Wistar大鼠随机分成对照组(9只)、非IP(NIP)组(45只)和IP组(45只)。大脑中动脉线栓法建立局灶性IP模型,NIP组以假手术代替预缺血,分别在IP后1、3、7、14、21 d予以缺血2 h、再灌注22 h,对照组2次均为假手术。大鼠处死前给予神经功能缺损评分(NDS),采用TTC染色测定脑梗死体积,免疫组化染色法检测脑组织VEGF、Ang-1蛋白表达。结果对照组大鼠脑组织无梗死灶,NDS为0。IP 1、3、7 d亚组NDS和梗死体积显著小于NIP组1、3、7 d亚组(P<0.01～0.05),其中IP 3 d亚组NDS最低,梗死体积最小。对照组脑组织少量VEGF蛋白表达,IP 1、3、7 d亚组VEGF蛋白表达显著高于NIP 1、3、7 d亚组(均P<0.05),其中IP 3 d亚组VEGF蛋白表达最高。各组脑组织均有一定程度Ang-1蛋白表达,IP 7 d亚组Ang-1蛋白表达高于NIP 7 d亚组(P<0.05)。结论 IP 1～7 d内脑组织VEGF、Ang-1表达增加,对脑缺血耐受的产生具有重要作用。     

依达拉奉对脑缺血再灌注大鼠血管内皮生长因子和血管生成素-1的影响

目的探索依达拉奉对脑缺血再灌注大鼠的血管内皮生长因子(VEGF)和血管生成素-1(Ang-1)的影响。方法采用大脑中动脉闭塞法建立大鼠脑缺血再灌注模型,将雄性SD大鼠随机分为3组:假手术组、模型组和依达拉奉组,每组19只。HE染色观察梗死灶周围的病理改变,透射电镜下观察梗死灶周围脑组织的毛细血管内皮形态变化,激光多普勒微循环血流仪监测微循环血流量,采用免疫组化检测脑组织VEGF和Ang-1的表达。结果依达拉奉组的组织病理损伤和血管内皮细胞损伤要轻于模型组,微循环血流量(47.32±6.58)明显高于模型组(40.51±7.96)(P0.05)。模型组大脑组织的VEGF和Ang-1[(0.124±0.021),(0.099±0.014)]均明显高于假手术组[(0.118±0.018),(0.095±0.016)](均P0.05),而依达拉奉组大脑组织的VEGF和Ang-1[(0.147±0.019),(0.124±0.021)]均明显高于模型组[(0.136±0.023),(0.118±0.018)](均P0.05)。结论依达拉奉能上调缺血再灌注大鼠脑组织的VEGF和Ang-1来保护梗死灶周围毛细血管的内皮细胞。     

Alzheimer病患者外周血淋巴细胞周期分布及细胞外信号调节激酶1/2/哺乳动物雷帕霉素靶蛋白通路的改变及意义

目的研究Alzheimer病(AD)患者外周血淋巴细胞周期分布及上游调控通路细胞外信号调节激酶1/2(ERK1/2)/哺乳动物雷帕霉素靶蛋白(m TOR)的改变及意义。方法对30例AD患者(AD组)用流式细胞分析仪检测外周血淋巴细胞周期分布,用Western Blot法检测淋巴细胞ERK1/2/m TOR通路和周期素依赖性蛋白激酶抑制剂p21的表达,并与帕金森病(PD)患者(PD组)及正常对照者(NC组)进行比较。分析AD组细胞周期相关蛋白与简易精神状态检查(MMSE)量表评分、病程的关系。结果与NC组相比,AD组外周淋巴细胞分布于G1期的比例(G1%)显著下降而S%显著升高(均P0.01);淋巴细胞中p-ERK1/2、pm TOR和p21水平显著下降(均P0.01)。与NC组相比,PD组ERK1/2、p-ERK1/2、m TOR、p-m TOR、p21的差异均无统计学意义。AD组外周血淋巴细胞p-m TOR水平与MMSE评分呈正相关(r=0.592,P=0.018),与病程呈负相关(r=-0.558,P=0.025)。结论 AD患者外周血淋巴细胞周期分布存在异常,淋巴细胞的pERK1/2、p-m TOR和p21水平显著下降,并且病情越重、病程越长者p-m TOR水平下降越明显。外周血淋巴细胞p-ERK1/2、p-m TOR和p21有可能成为AD诊断及病情评价的外周标志物。     

Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases

Microglia are activated in response to a number of different pathological states within the CNS including injury, ischemia, and infection. Microglial activation results in their production of pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α. While release of these factors is typically intended to prevent further damage to CNS tissue, they may also be toxic to neurons and other glial cells. Mounting evidence indicates that chronic microglial activation may also contribute to the development and progression of neurodegenerative disorders. Unfortunately, determining the role of pro-inflammatory cytokines in these disorders has been complicated by their dual roles in neuroprotection and neurodegeneration. The purpose of this review is to summarize current understanding of the involvement of cytokines in neurodegenerative disorders and their potential signaling mechanisms in this context. Taken together, recent findings suggest that microglial activation and pro-inflammatory cytokines merit interest as targets in the treatment of neurodegenerative disorders.     

Cellular signaling roles of TGFβ, TNFα and βAPP in brain injury responses and Alzheimer's disease

β-Amyloid precursor protein (βAPP), transforming growth factor β (TGFβ), and tumor necrosis factor-α (TNFα) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGFβ and TNFα in characteristic ways when the brain is injured. TGFβ, TNFα and secreted forms of βAPP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNFα and the fibrillogenic amyloid β-peptide (Aβ) derivative of βAPP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGFβ, TNFα or βAPP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGFβ (serine/threonine kinase cascades), TNFα (p55 receptor linked to a sphingomyelin-ceramide-NFκB pathway), and secreted forms of βAPP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K⁺ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.