Scatter factor/hepatocyte growth factor gene transfer increases rat blood–glioma barrier permeability

Malignant gliomas are associated with a dysfunctional blood–tumor barrier (BTB) that causes substantial morbidity. Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional growth factor that correlates with glioma malignancy and has several biological properties that suggest a role in enhancing blood–glioma barrier permeability. In this study, we examined the effects of glioma cell SF/HGF expression on BTB permeability to horseradish peroxidase (HRP). Fischer 344 rats bearing intrastriatal 9L tumors engineered to secrete SF/HGF (9L-SF) and SF/HGF-negative control tumors (9L-neo) received intracardiac injections of HRP and were rapidly decapitated. Densitometric analysis of brain sections reacted with diaminobenzidine showed significantly greater extravascular HRP surrounding SF/HGF-secreting tumors than 9L-neo tumors of comparable size (p<0.05). HRP enzymatic activity associated with striata containing SF/HGF-expressing tumors was 1.6-fold greater than that of striata containing control tumors (p<0.05). Northern analysis showed that expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) did not differ between 9L-neo and 9L-SF tumors. These data demonstrate that SF/HGF expression by intracerebral glial tumors can enhance BTB permeability independent of changes in VEGF/VPF expression.     

Effects of insulin combined with idebenone on blood–brain barrier permeability in diabetic rats

This study investigates the effect of insulin combined with idebenone on blood–brain barrier (BBB) permeability in experimental streptozotocin‐induced diabetic rats as well as the underlying mechanisms. With a diabetic rat model, we show that insulin and idebenone normalize body weight and water intake and restore BBB permeability and that their combination displays a synergistic effect. The results from transmission electron microscopy show that the combination of insulin and idebenone significantly closed the tight junction (TJ) in diabetic rats. The results from Western blotting in diabetic rats show that the upregulation of TJ‐associated proteins occludin, and zonula occludens (ZO)‐1 caused by the combination of insulin and idebenone is more remarkable than that with either agent alone. In addition, the activations of reactive oxygen species (ROS) and advanced glycation end products (AGEs) and the expression levels of receptors for advanced glycation end‐products (RAGE) and nuclear factor‐κB (NF‐κB) were significantly decreased after treatment with insulin and idebenone in diabetic rats. These results suggest that the combination of insulin and idebenone could decrease the BBB permeability in diabetic rats by upregulating the expression of occludin, claudin‐5, and ZO‐1 and that the ROS/AGE/RAGE/NF‐κB signal pathway might be involved in the process. © 2014 Wiley Periodicals, Inc.     

Overproduction of vascular endothelial growth factor/vascular permeability factor is causative in crow–fukase (POEMS) syndrome

Crow–Fukase or POEMS syndrome of polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes is a rare multisystem disorder of obscure pathogenesis that is associated with microangiopathy, neovascularization, and accelerated vasopermeability. We examined the levels of the vascular endothelial growth factor/vascular permeability factor (VEGF) in the serum and cerebrospinal fluid (CSF) from 10 patients with this syndrome. Serum VEGF levels were about 15–30 times those in control subjects or patients with Guillain–Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and other neurological disorders. The CSF VEGF levels, however, were similar to those found in GBS and CIDP. Elevated VEGF levels in the serum decreased in 7 patients with Crow–Fukase syndrome after conventional therapy. The principal isoform of VEGF in Crow–Fukase syndrome was VEGF165. Elevated VEGF was independent of M-protein. Our results suggest that the overproduction of VEGF is important in the pathogenesis of this disorder. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1390–1397, 1998     

Correlation of vascular endothelial growth factor to permeability of blood-brain barrier and brain edema during high-altitude exposure

BACKGROUND： Many studies have evaluated the role of vascular endothelial growth factor （VEGF） in traumatic brain edema and hemorrhagic brain edema. OBJECTIVE： To observe the effects of VEGF expression on permeability of the blood-brain barrier （BBB） during high-altitude and hypoxia exposure, and to investigate the correlation between VEGF expression and BBB permeability with regard to Evans blue staining and brain edema during high-altitude exposure. DESIGN, TIME AND SETTING： The randomized, controlled, animal study was performed at the Tanggula Etape, Central Laboratory of Chengdu Medical College, and Central Laboratory of General Hospital of Chengdu Military Area Command of Chinese PLA, China, from July 2003 to November 2004. MATERIALS： Quantitative RT-PCR kit （Sigma, USA）, VEGF ELISA kit （Biosource, USA）, and Evans blue （Jingchun, China） were acquired for this study. METHODS： A total of 180 Wistar rats were equally and randomly assigned to 15 groups： low-altitude （500 m）, middle-altitude （2 880 m）, high-altitude （4 200 m）, super-high-altitude （5 000 m）, 1,3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 days of super high-altitude exposure. Wistar rats were exposed to various altitude gradients to establish a hypoxia model. MAIN OUTCOME MEASURES： Brain water content was calculated according to the wet-to-dry weight ratio. BBB permeability to Evans blue was determined by colorimetric method. VEGF mRNA and protein levels in brain tissues were detected using RT-PCR and double-antibody sandwich ELISA. RESULTS： Brain water content, BBB permeability to Evans blue, and VEGF mRNA and protein levels in brain tissues increased with increasing altitude and prolonged exposure to altitude. The greatest increase was determined on day 9 upon ascending 5 000 m. Simultaneously, VEGF expression positively correlated to BBB permeability of Evans blue and brain water content （r = 0.975, 0.917, P〈 0.01）. CONCLUSION： Increased VEGF protein and mRNA expression was responsible for increased BBB permeability, which may be an important mechanism underlying brain edema during high-altitude exposure.     

Permeability of the blood–brain barrier to neurotrophins

To evaluate the feasibility of applying blood-borne neurotrophins to promote normal function of the central nervous system (CNS) and to rescue neuronal degeneration, we characterized the permeability of the blood–brain barrier (BBB) to neurotrophins. We report here that some members of the neurotrophin family (NGF, βNGF, NT3, and NT5) can cross the BBB of mice in vivo to arrive at the brain parenchyma. BBB permeability differed among individual neurotrophins in that NGF had the fastest influx rate (K_i) and NT3 the slowest, and that the entry rate of NGF was twice that of its smaller bioactive subunit βNGF. BBB permeability also differed at various CNS regions in that the cervical spinal cord had the greatest rate of influx, whereas brain had the lowest. Saturability of influx was suggested by self-inhibition studies for NT3 in vivo, and for NGF in an in situ brain perfusion system, indicating the presence of saturable transport systems. The results suggest that peripheral administration of neurotrophins could have therapeutic effects within the CNS.     

Regulatory effect of vascular endothelial growth factor on blood spinal cord barrier in presyrinx state of experimental syringomyelia

BACKGROUND:Vascular endothelial growth factor (VEGF) is able to regulate blood spinal cord barrier function as well as influence neovascularization and cause edema. OBJECTIVE: Through establishment of a rabbit model of syringomyelia,to explore the correlation between VEGF protein and mRNA expressions and function of blood spinal cord barrier and edema degree of spinal cord in presyrinx state. DESIGN,TIME AND SETTING: Randomized controlled animal study was performed in the Tumor Institute of the Fourth Hospi...     

Regulatory effect of vascular endothelial growth factor on blood spinal cord barrier in presyrinx state of experimental syringomyelia☆

BACKGROUND： Vascular endothelial growth factor （VEGF） is able to regulate blood spinal cord barrier function as well as influence neovascularization and cause edema. OBJECTIVE： Through establishment of a rabbit model of syringomyelia, to explore the correlation between VEGF protein and mRNA expressions and function of blood spinal cord barrier and edema degree of spinal cord in presyrinx state. DESIGN, TIME AND SETTING： Randomized controlled animal study was performed in the Tumor Institute of the Fourth Hospital, Hebei Medical University from January to June 2007. MATERIALS： Atotal of 0.6 mL Kaolin solution （250 g/L, 37℃） was injected into the cisterna magna of 40 rabbits in the kaolin group to establish syringomyelia models. Goat anti-rabbit VEGF monoclonal antibody was provided by DIACLONE Company, USA; RT-PCR related reagents were provided by Huamei Bioengineering Co., Ltd., Beijing. METHODS： Sixty Chinese white rabbits were divided randomly into two groups： Kaolin group （n = 40） and control group （n = 20）. Physiological saline （0.6 mL at 37℃） was injected in rabbits of control group. On days 1,3, 7, 14 and 21 after kaolin injection, cervical cords samples were harvested after sacrifice of animal. MAIN OUTCOME MEASURES： VEGF protein and mRNA expressions were detected by immunohistochemistry and RT-PCR on days 1, 3, 7, 14, and 21 after kaolin injection. A quantitative analysis of blood spinal cord barrier function was performed by Evans blue technique. Water content of the spinal cord was measured by dry-wet weight technique. The correlation between the expression of VEGF protein and mRNA and the function of blood spinal cord barrier in the upper cervical cord of the presyrinx state was analyzed by linear correlation analysis. RESULTS： The water content and Evans blue content increased in the kaolin group on days 1 and 3 postoperatively compared with the control group （F = 7.387, 61.35, P 〈 0.05, 0.01）, and reached a peak on day 7 （F = 135.94, 528.35, P 〈 0.01?     

Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins

The outcome of stroke is greatly influenced by the state of the blood–brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.     

纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子表达的影响

目的观察纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子(VEGF)转录及蛋白水平表达的影响。方法大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过Real-time PCR检测VEGF转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的VEGF水平。结果纤维蛋白可以特异性诱导大鼠脑血管内皮细胞表达VEGF;加入不同浓度的纤维蛋白(0.03mg/ml、0.1mg/ml、0.3mg/ml和1.0mg/ml),24h后,1.0mg/ml纤维蛋白组的培养基VEGF水平显著增高(P<0.001);1.0mg/ml纤维蛋白与大鼠脑血管内皮细胞分别培养0、2、4、8、24、48h,VEGF浓度在共培养2h已经升高,8h时显著升高,在24h时仍然保持在显著升高表达水平(P<0.005),48h有所下降;Real-time PCR结果提示,大鼠脑血管内皮细胞中VEGF mRNA的上调呈现出剂量和时间依赖性增加。结论纤维蛋白可以上调大鼠血管内皮细胞中的VEGF。     

Improvement of cold injury‐induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor‐A

Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ET_B receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ET_B antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)‐A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5–6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF‐A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF‐A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 μg) or IRL‐2500 (10 μg) (selective ET_B antagonists) attenuated brain edema and disruption of the blood–brain barrier after cold injury. BQ788 and IRL‐2500 reversed the cold injury‐induced increases in MMP9 and VEGF‐A expression. The induction of reactive astrocytes producing MMP9 and VEGF‐A in the damaged cerebrum was attenuated by BQ788 and IRL‐2500. These results suggest that attenuations of astrocytic MMP9 and VEGF‐A expression by ET_B antagonists may be involved in the amelioration of vasogenic brain edema.     

Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the vasculature of oligodendrogliomas

C. Christov, H. Adle-Biassette, C. Le Guerinel, S. Natchev and R. K. Gherardi (1998) Neuropathology and Applied Neurobiology 24, 29–35 Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the vasculature of oligodendrogliomas Vascular endothelial growth factor (VEGF) appears to be implicated in tumour angiogenesis. In the present study immunohistochemical expression of VEGF was evaluated in 34 oligodendrogliomas (13 grade II, 21 grade III [WHO]). VEGF immunoreactivity was found in 31 of 34 cases. Expression of VEGF was observed in endothelial cells and some vascular smooth muscle cells, but not in neoplastic oligodendrocytes. Vessel counts, percentages of VEGF-positive vessels and vessels with vascular endothelial proliferation were assessed. The degree of VEGF labelling and vascular-endothelial proliferation in each vessel were evaluated using a 3 degree intensity score. Expression of VEGF was higher in grade III than in grade II oligodendrogliomas as assessed by percentage of VEGF positive vessels (55.8 ± 29.2% vs 17.0 ± 19.0% [P < 0.001]) and by VEGF immunostaining intensity (1.90 ± 0.60 vs 0.90 ± 0.40 [P < 0.001]). VEGF expression did not correlate with vessel density. Intensity of VEGF expression correlated positively with that of vascular-endothelial proliferation in grade III tumours (r=+0.47 [P < 0.05]). The percentage of VEGF positive vessels showed some degree of positive correlation with the percentage of vessels showing vascular-endothelial proliferation (r=+408 [P < 0.10]). Within individual grade III tumours 67.5 ± 29.6% of all vessels with vascular-endothelial proliferation were VEGF-positive and 31.0 ± 20.5% of all VEGF-positive vessels showed no evidence of vascular-endothelial proliferation. We conclude that (i) expression of VEGF is observed in the vasculature of oligodendrogliomas; (ii) marked expression of VEGF is observed in grade III oligodendrogliomas; (iii) VEGF may be one of the interrelated causative stimuli acting in concert to induce vascular-endothelial proliferation.     

Imatinib preserves blood–brain barrier integrity following experimental subarachnoid hemorrhage in rats

Blood–brain barrier (BBB) disruption and consequent edema formation contribute to the development of early brain injury following subarachnoid hemorrhage (SAH). Various cerebrovascular insults result in increased platelet‐derived growth factor receptor (PDGFR)‐α stimulation, which has been linked to BBB breakdown and edema formation. This study examines whether imatinib, a PDGFR inhibitor, can preserve BBB integrity in a rat endovascular perforation SAH model. Imatinib (40 or 120 mg/kg) or a vehicle was administered intraperitoneally at 1 hr after SAH induction. BBB leakage, brain edema, and neurological deficits were evaluated. Total and phosphorylated protein expressions of PDGFR‐α, c‐Src, c‐Jun N‐terminal kinase (JNK), and c‐Jun were measured, and enzymatic activities of matrix metalloproteinase (MMP)?2 and MMP‐9 were determined in the injured brain. Imatinib treatment significantly ameliorated BBB leakage and edema formation 24 hr after SAH, which was paralleled by improved neurological functions. Decreased brain expressions of phosphorylated PDGFR‐α, c‐Src, JNK, and c‐Jun as well as reduced MMP‐9 activities were found in treated animals. PDGFR‐α inhibition preserved BBB integrity following experimental SAH; however, the protective mechanisms remain to be elucidated. Targeting PDGFR‐α signaling might be advantageous to ameliorate early brain injury following SAH. © 2014 Wiley Periodicals, Inc.     

Inhibition of calcium/calmodulin‐dependent protein kinase kinase (CaMKK) exacerbates impairment of endothelial cell and blood–brain barrier after stroke

Brain microvascular endothelial cells play an essential role in maintaining blood–brain barrier (BBB) integrity, and disruption of the BBB aggravates the ischemic injury. CaMKK (α and β) is a major kinase activated by elevated intracellular calcium. Previously, we demonstrated that inhibition of CaMKK exacerbated outcomes, conversely, overexpression reduced brain injury after stroke in mice. Interestingly, CaMKK has been shown to activate a key endothelial protector, sirtuin 1 (SIRT1). We hypothesized that CaMKK protects brain endothelial cells via SIRT1 activation after stroke. In this study, Oxygen‐Glucose Deprivation (OGD) was performed in human brain microvascular endothelial cells. Stroke was induced by middle cerebral artery occlusion (MCAO) in male mice. Knockdown of CaMKK β using siRNA increased cell death following OGD. Inhibition of CaMKK β by STO‐609 significantly and selectively down‐regulated levels of phosphorylated SIRT1 after OGD. Changes in the downstream targets of SIRT1 were observed following STO‐609 treatment. The effect of STO‐609 on cell viability after OGD was absent, when SIRT1 was concurrently inhibited. We also demonstrated that STO‐609 increased endothelial expression of the pro‐inflammatory proteins ICAM‐1 and VCAM‐1 and inhibition of CaMKK exacerbated OGD‐induced leukocyte‐endothelial adhesion. Finally, intracerebroventricular injection of STO‐609 exacerbated endothelial apoptosis and reduced BBB integrity after 24‐hr reperfusion following MCAO in vivo. Collectively, these results demonstrated that CaMKK inhibition reduced endothelial cell viability, exacerbated inflammatory responses and aggravated BBB impairment after ischemia. CaMKK activation may attenuate ischemic brain injury via protection of the microvascular system and a reduction in the infiltration of pro‐inflammatory factors.     

Post-ischemic leakiness of the blood–brain barrier: A quantitative and systematic assessment by Patlak plots

The Patlak plot analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) allows estimation of blood–brain barrier (BBB) leakage following temporary focal cerebral ischemia. Thus far, a systematic and quantitative in vivo evaluation of post-ischemic BBB leakage is lacking. Here, using DCE-MRI and the Patlak plot method, we quantitatively assessed BBB leakage in rats at the following time-points after reperfusion: 25 min, 2, 4, 6, 12, 18, 24, 36, 48, and 72 h, and 1, 2, 3, 4, and 5 weeks. Sham-operated animals served as controls. Data collected for each time-point were: the blood-to-brain transfer rate constant (K_i) of the contrast agent gadolinium, distribution volume (V_p), ischemic lesion volume, and apparent diffusion coefficient (ADC) values. Compared to controls, K_i, measured at all time-points, except for 5 weeks, appeared significantly different (p < 0.001). At several time-points (25 min, 48 and 72 h, 4 and 5 weeks), V_p was similar compared to that of controls, but for the remaining groups the difference was significant (p < 0.001). Analyzing the relationship of K_i values to time-points, we observed a trend towards a decrease over time (r = − 0.61, p = 0.014). Both ADC values (r = − 0.58, p = 0.02) and ischemic lesion volumes (r = 0.75, p = 0.0015) correlated with K_i values. These results suggest that after ischemia–reperfusion in rats, BBB leakage is continuous during a 4-week period. Its magnitude diminishes over time and correlates with severity and extent of ischemic injury.     

步长脑心通对血管性痴呆大鼠血管内皮细胞生长因子作用的影响

目的:观察步长脑心通对VD模型海马区血管内皮细胞生长因子(VEGF)的影响。方法:将50只大鼠随机分为正常组、假手术组、模型组、西药(喜德镇)组及步长脑心通组,建立实验动物模型。分别于制模后、30天后应用免疫组化法分析大鼠海马部位VEGF分布情况。结果:步长脑心通组大鼠海马组织内VEGF明显增加。结论:步长脑心通能明显增强VD模型大鼠海马组织VEGF表达,从而减轻VD模型大鼠的缺血性损伤。     

N‐myc downstream‐regulated gene 2 protects blood–brain barrier integrity following cerebral ischemia

Disruption of the blood‐brain barrier (BBB) following cerebral ischemia is closely related to the infiltration of peripheral cells into the brain, progression of lesion formation, and clinical exacerbation. However, the mechanism that regulates BBB integrity, especially after permanent ischemia, remains unclear. Here, we present evidence that astrocytic N‐myc downstream‐regulated gene 2 (NDRG2), a differentiation‐ and stress‐associated molecule, may function as a modulator of BBB permeability following ischemic stroke, using a mouse model of permanent cerebral ischemia. Immunohistological analysis showed that the expression of NDRG2 increases dominantly in astrocytes following permanent middle cerebral artery occlusion (MCAO). Genetic deletion of Ndrg2 exhibited enhanced levels of infarct volume and accumulation of immune cells into the ipsilateral brain hemisphere following ischemia. Extravasation of serum proteins including fibrinogen and immunoglobulin, after MCAO, was enhanced at the ischemic core and perivascular region of the peri‐infarct area in the ipsilateral cortex of Ndrg2‐deficient mice. Furthermore, the expression of matrix metalloproteinases (MMPs) after MCAO markedly increased in Ndrg2^?/? mice. In culture, expression and secretion of MMP‐3 was increased in Ndrg2^?/? astrocytes, and this increase was reversed by adenovirus‐mediated re‐expression of NDRG2. These findings suggest that NDRG2, expressed in astrocytes, may play a critical role in the regulation of BBB permeability and immune cell infiltration through the modulation of MMP expression following cerebral ischemia.     

Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood–brain barrier

The uptake of biotin and the closely related biocytin was characterized in primary cultures of calf brain microvessel endothelial (CBME) cells. Biotin uptake was found to be Na⁺-gradient dependent and independent of changes in the membrane potential. Concentration dependence revealed a single saturation mechanism with a K_m of 47 μM and a V_max of 101 pmol/min/mg. Inhibition studies demonstrated dependence on metabolic energy and the necessity for a free carboxyl group for transport activity. The anticonvulsants primidone and carbamazepine had no inhibitory effect. Biotin uptake into CBME cells is a secondary active, electroneutral, saturable and specific process. Biocytin which accumulates in biotinidase deficiency, a human congenital disorder, did not inhibit biotin uptake and was not transported into these cells. The presence of human serum with normal biotinidase activity significantly reduced biotin uptake by about 50%. Further, added biocytin was hydrolyzed to biotin, which accumulated intracellularly but to a lesser extent than added free biotin. Biotin uptake after addition of plasma of biotinidase-deficient patients was not different from that in the presence of normal serum. These results indicate that the absence of biotinidase activity in serum does not reduce blood–brain barrier transport of biotin.