VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.

VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a "survival," rather than an "angiogenic" factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.

VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

Localization of plasminogen in mouse hippocampus, cerebral cortex, and hypothalamus.

Although the tissue plasminogen activator/plasminogen system contributes to numerous brain functions, such as learning, memory, and anxiety behavior, little attention has as yet been given to the localization of plasminogen in the brain. We have investigated the localization of plasminogen in the adult mouse brain by using immunohistochemistry. In the hippocampus, plasminogen immunoreactivity was seen in the pyramidal cell layer as numerous punctate structures in neuronal somata. An electron-microscopic study further demonstrated that the plasminogen-immunoreactive punctate structures represented secretory vesicles and/or vesicle clusters. In the cerebral cortex, plasminogen immunoreactivity was evident in the somata of the layer II/III and V neurons. A quantitative analysis revealed that parvalbumin (PV)-positive neurons had more plasminogen-immunoreactive puncta compared with those of PV-negative neurons in the hippocampus and cerebral cortex. Plasminogen immunoreactivity was present throughout the hypothalamus, being particularly prominent in the neuronal somata of the organum vasculosum laminae terminalis, ventromedial preoptic nucleus, supraoptic nucleus, subfornical organ, medial part of the paraventricular nucleus (PVN), posterior part of the PVN, and arcuate hypothalamic nucleus. Thus, plasminogen is highly expressed in specific populations of hippocampal, cortical, and hypothalamic neurons, and plasminogen-containing vesicles are mainly observed at neuronal somata.

Tissue-type plasminogen activator (t-PA) induces stromelysin-1 (MMP-3) in endothelial cells through activation of lipoprotein receptor-related protein.

Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it increases the risk of intracranial bleeding (ICB). Previously, we have shown in a mouse stroke model that stromelysin-1 (matrix metalloproteinase-3 [MMP-3]) induced in endothelial cells was critical for ICB induced by t-PA. In the present study, using bEnd.3 cells, a mouse brain-derived endothelial cell line, we showed that MMP-3 was induced by both ischemic stress and t-PA treatment. This induction by t-PA was prevented by inhibition either of low-density lipoprotein receptor-related protein (LRP) or of nuclear factor-kappaB activation. LRP was up-regulated by ischemic stress, both in bEnd.3 cells in vitro and in endothelial cells at the ischemic damage area in the mouse stroke model. Furthermore, inhibition of LRP suppressed both MMP-3 induction in endothelial cells and the increase in ICB by t-PA treatment after stroke. These findings indicate that t-PA deteriorates ICB via MMP-3 induction in endothelial cells, which is regulated through the LRP/nuclear factor-kappaB pathway.

Novel situations of endothelial injury in stroke--mechanisms of stroke and strategy of drug development: intracranial bleeding associated with the treatment of ischemic stroke: thrombolytic treatment of ischemia-affected endothelial cells with tissue-type plasminogen activator.

Previous studies have shown that the risk of intracranial hemorrhage (ICH) associated with the treatment of ischemic stroke is mainly attributable to antithrombotic agents. On the basis of clinical trials, only tissue-type plasminogen activator (t-PA) has been approved for treating acute ischemic strokes, but delayed treatment with t-PA is associated with the risk of cerebral hemorrhagic transformation of ischemic stroke. t-PA converts plasminogen to plasmin, which participates primarily in clot lysis via fibrin degradation and, to some extent, in tissue remodeling via degradation of various extracellular matrix proteins, either directly or via activation of matrix metalloproteinases (MMPs). MMPs mediate the pathogenesis of ischemic-stroke-associated ICH by causing the disruption of vasculature. In particular, the binding of t-PA with one of its receptors leads to the activation of low-density lipoprotein receptor-related protein (LRP), which in turn results in the release of MMP-3 by endothelial cells. LRP production is reported to be upregulated in endothelial cells exposed to ischemia, and elevated LRP levels have been implicated in the increased ICH risk associated with delayed t-PA treatment. This implies that the t-PA / LRP / MMP-3 pathway may be a suitable target for developing strategies to improve the therapeutic efficacy of t-PA in acute ischemic stroke.

Profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus.

Fibrinolytic system impairment contributes to the development of thrombotic disease such as cardiovascular disease and stroke. Therefore, an agent that increases fibrinolytic activity may be useful for the prevention of these diseases. In this study, to explore novel profibrinolytic agents, we examined the profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus in vitro and in vivo. Enzamin directly enhanced plasmin activity generated by tissue-type plasminogen activator (t-PA) by twofold but not by urokinase-type plasminogen activator (u-PA) in vitro, which was measured employing both the chromogenic substrate H-D: -Val-Leu-Lys-pNA (S-2251) and fibrin plate. Enzamin also increased plasmin activity generated by t-PA in the cell lysate and culture medium of endothelial cells, measured by fibrin zymography. Furthermore, the oral administration of a 1% concentration of Enzamin increased plasmin activity generated by t-PA by 1.7-fold but not by u-PA in the euglobulin fraction of mouse plasma. In conclusion, Enzamin has a unique ability to enhance the fibrinolytic activity through an increase in endogenous plasmin activity generated by t-PA released from endothelial cells, and may be a beneficial supplement for the prevention of thrombotic episodes.

Repair of brain damage size and recovery of neurological dysfunction after ischemic stroke are different between strains in mice: evaluation using a novel ischemic stroke model.

In the current study, we established a novel murine ischemic brain damage model using a photochemical reaction to evaluate the recovery of neurological dysfunction and brain repair reactions. In this model, reproducible damage was induced in the frontal lobe of the cortex, which was accompanied by neurological dysfunction. Sequential changes in damage size, microglial accumulation, astrocyte activation, and neurological dysfunction were studied in C57BL/6J and BALB/c mouse strains. Although the initial size of damage was comparable in both strains, the extent of damage was later reduced to a greater extent in C57BL/6J mice than that in BALB/c mice. In addition, C57BL/6J mice showed later edema clearance until day 7, less microglial accumulation, and relatively more astrocyte activation on day 7. Neurologic dysfunction was evaluated by three behavioral tests: the von Frey test, the balance beam test, and the tail suspension test. The behavioral abnormalities evaluated by these tests were remarkable following the induction of damage and recovered by day 21 in both strains. However, the abnormalities were more prominent and the recovery was later in C57BL/6J mice. These findings demonstrate that our novel ischemic stroke model is useful for evaluating brain repair reactions and the recovery of neurological dysfunction in mice with different genetic backgrounds. In addition, we found that both the brain repair reactions and the recovery of neurological dysfunction after comparable ischemic brain damage varied between strains; in that, they both occurred later in C57BL/6J mice.

Protective role of reactive astrocytes in brain ischemia.

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP(-/-)Vim(-/-) mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP(-/-)Vim(-/-) than in wild-type (WT) mice; GFAP(-/-), Vim(-/-) and WT mice had the same infarct volume. Endothelin B receptor (ET(B)R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP(-/-)Vim(-/-) astrocytes. In WT astrocytes, ET(B)R colocalized extensively with bundles of IFs. GFAP(-/-)Vim(-/-) astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP(-/-)Vim(-/-) than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET(B)R-mediated control of gap junctions, and PAI-1 expression.

Urokinase-type plasminogen activator receptor (uPAR) augments brain damage in a murine model of ischemic stroke.

Urokinase-type plasminogen activator receptor (uPAR) is a key component of the plasminogen activation system at the cell surface. Recent studies showed that uPAR is expressed in the ischemic damaged brain, suggesting its involvement in brain damage. In this study, we evaluated the role of uPAR in ischemic brain damage induced by permanent middle cerebral artery (MCA) occlusion in mice with genetic deficiency of uPAR (uPAR(-/-)) or of uPA (uPA(-/-)). Brain damage at 3 days was smaller in uPAR(-/-) mice (4.5+/-1.0mm(3)) than in littermate wild-type mice (uPAR(+/+)) (9.1+/-1.8mm(3), p<0.05), whereas it was comparable in uPA(-/-) (8.0+/-4.1mm(3)) and uPA(+/+) (6.9+/-2.6mm(3)) mice. uPAR expression was upregulated in the ipsilateral cerebral cortex within 12h, and remained elevated for up to 3 days. At 1 or 2 days after MCA occlusion, uPAR expression was selectively localized in vessels at the border of the damaged area. These findings suggest that uPAR expressed by endothelial cells augments the ischemic brain damage via a uPA-independent mechanism.

Prothrombotic effect of Rofecoxib in a murine venous thrombosis model.

The potential prothrombotic effect of the cyclooxygenase-2 (COX-2) inhibitor Rofecoxib (Vioxx) was investigated using murine thrombosis models. In a jugular vein thrombosis model (photochemically induced injury) in lean wild-type mice, Rofecoxib treatment for 4 weeks induced a mild prothrombotic tendency, as indicated by a shorter occlusion time as compared to placebo (median of 12 min versus 36 min; p < 0.05). Thrombus size was somewhat, but not significantly, enhanced after Rofecoxib treatment. In a femoral artery thrombosis model (FeCl3 induced injury) Rofecoxib did not cause an enhanced thrombotic tendency in mice with nutritionally induced or genetically determined (ob/ob) obesity. The occlusion time was comparable for obese wild-type mice with (8.8+/-0.7 min) or without (7.8+/-2.1 min) Rofecoxib treatment, as well as for ob/ob mice (8.5+/-0.7 min versus 6.8+/-3.0 min). Thus, an enhanced prothrombotic effect of Rofecoxib was detected when using a venous thrombosis model in lean mice, but not when using an arterial thrombosis model in obese mice.

Obesity promotes injury induced femoral artery thrombosis in mice.

An FeCl(3) induced femoral arterial thrombosis model was applied to lean (47+/-1.4 g) and obese (64+/-1.7 g) mice (Swiss genetic background) in order to study the relation between obesity and thrombotic risk. As compared to lean mice, obese mice showed a significantly shorter occlusion time (9.9+/-1.0 min versus 13+/-0.5 min; p=0.04) and lower total blood flow (37+/-7.3% versus 69+/-6.7%; p=0.008). A significant negative correlation was observed between body weight and both occlusion time (r=-0.57; p=0.014) and blood flow (r=-0.57; p=0.028). Analysis of the coagulation profile revealed significantly higher levels of plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex, Factor V activity and combined Factors II/VII/X activity, and moderately elevated Factor VIII activity in obese mice. The degree of arterial damage and the thrombus extension were, however, not significantly different. A significant positive correlation was observed between body weight and either PAI-1 (r=0.63; p=0.003), Factors II/VII/X levels (r=0.80; p<0.0001) or Factor V levels (r=0.65; p=0.003). Thus, this injury induced femoral artery thrombosis model in mice establishes experimentally a correlation between obesity and prothrombotic tendency.

Increase in blood-brain barrier permeability does not directly induce neuronal death but may accelerate ischemic neuronal damage.

It is observed that the increase in blood-brain barrier (BBB) permeability (BBBP) is associated with ischemic stroke and thought to trigger neuronal damage and deteriorate ischemic infarction, even though there is no experimental proof. Here, we investigated the effect of BBBP increase on brain damage, using a combination of photochemically-induced thrombotic brain damage (PIT-BD) model, a focal brain ischemic model, and transient bilateral carotid artery occlusion model (CAO, a whole brain ischemic model), in mice. In PIT-BD, BBBP increased in the region surrounding the ischemic damage from 4 h till 24 h with a peak at 8 h. On day 4, the damaged did not expand to the region with BBBP increase in mice with PIT-BD alone or with 30 min CAO at 1 h before PIT-BD, but expanded in mice with 30 min CAO at 3.5 h after PIT-BD. This expansion was paralleled with the increase in the number of apoptotic cells. These findings indicate that increase in BBBP does not cause direct neuronal death, but it facilitates ischemic neuronal loss, which was attributed, at least partially, to acceleration of apoptotic cell death.

Factor V Leiden mutation is associated with enhanced arterial thrombotic tendency in lean but not in obese mice.

The homozygous factor V Leiden mutation is associated with enhanced venous thrombotic risk. Obesity is a major risk factor for development of thrombotic cardiovascular disease. It was the objective of this study to investigate whether obesity affects the thrombotic risk associated with the mutation. Male mice with homozygous factor V Leiden mutation (Arg 504 to Gln) (FVQ/Q) and corresponding wild-type (WT) mice were kept on a standard fat diet (SFD) or high fat diet (HFD) for 14 weeks, and femoral artery thrombosis was induced by FeCl3 treatment. As compared to SFD, HFD feeding for 14 weeks resulted in significantly higher body weight and fat mass associated with adipocyte hypertrophy, which were, however, similar for both genotypes. In the FeCl3-induced arterial thrombosis model, FVQ/Q mice kept on SFD had a 40% shorter occlusion time (p = 0.015) and 40% lower blood flow (p = 0.03), as compared to WT mice. However, on HFD the occlusion time and blood flow were not significantly different for both genotypes. This finding could not be explained by differential changes of coagulation factors in either genotype fed on SFD or HFD. In conclusion, on SFD, but not on HFD, the factor V Leiden mutation is associated with enhanced thrombotic tendency after FeCl3 injury of the femoral artery, suggesting that in this model obesity rescues the increased thrombotic risk associated with the factor V Leiden mutation.

Delayed perfusion phenomenon in a rat stroke model at 1.5 T MR: an imaging sign parallel to spontaneous reperfusion and ischemic penumbra?

INTRODUCTION: Delayed perfusion (DP) sign at MR imaging was reported in stroke patients. We sought to experimentally elucidate its relation to spontaneous reperfusion and ischemic penumbra. METHODS: Stroke was induced by photothrombotic occlusion of middle cerebral artery in eight rats and studied up to 72 h using a 1.5 T MR scanner with T2 weighted imaging (T2WI), diffusion weighted imaging (DWI), and dynamic susceptibility contrast-enhanced perfusion weighted imaging (DSC-PWI). Relative signal intensity (rSI), relative lesion volume (rLV), relative cerebral blood flow (rCBF), PWI(rLV)-DWI(rLV) mismatch (penumbra) and DP(rLV) were quantified and correlated with neurological deficit score (NDS), triphenyl tetrazolium chloride (TTC) staining, microangiography (MA) and histopathology. RESULTS: The rSI and rLV characterized this stroke model on different MRI sequences and time points. DSC-PWI reproduced cortical DP in all rats, where rCBF evolved from 88.9% at 1 h through 64.9% at 6 h to 136.3% at 72 h. The PWI(rLV)-DWI(rLV) mismatch reached 10+/-5.4% at 1 h, remained positive through 12 h and decreased to -3.3+/-4.5% at 72 h. The incidence and rLV of the DP were well correlated with those of the penumbra (p<0.01, r(2)=0.85 and p<0.0001, r(2)=0.96, respectively). Shorter DP durations and more collateral arterioles occurred in rats without (n=4) than with (n=4) cortex involvement (p<0.05). Rats without cortex involvement tended to earlier reperfusion and a lower NDS. Microscopy confirmed MRI, MA and TTC findings. CONCLUSIONS: In this rat stroke model, we reproduced clinically observed DP on DSC-PWI, confirmed spontaneous reperfusion, and identified the penumbra extending to 12h post-ischemia, which appeared interrelated.

Rodent stroke induced by photochemical occlusion of proximal middle cerebral artery: evolution monitored with MR imaging and histopathology.

PURPOSE: To longitudinally investigate stroke in rats after photothrombotic occlusion of proximal middle cerebral artery (MCA) with magnetic resonance imaging (MRI) in correlation with histopathology. MATERIALS AND METHODS: Forty-two rats were subjected to photochemical MCA occlusion and MRI at 1.5T, and sacrificed in seven groups (n=6 each) at the following time points: 1, 3, 6 and 12h, and at day 1, 3 and 9. T2-weighted (T2WI) and diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) map was performed in all rats. Contrast-enhanced T1-weighted imaging (CE-T1WI) was compared to intravital staining with Evans blue in one group for assessing blood-brain barrier (BBB) integrity. The brain was stained histochemically with triphenyl tetrazolium chloride (TTC) and processed for pathological assessment. The evolutional changes of relative lesion volume, signal intensity (SI), and the BBB integrity on MRI with corresponding histopathology were evaluated. RESULTS: The ischemic lesion volume reached a maximum around 12h to day 1 as visualized successively by DWI, ADC map and T2WI, implicating the evolving pathology from cytotoxic edema through vasogenic edema to tissue death. The ADC of brain infarction underwent a significant reversion after 12h, reflecting the colliquative necrosis. On CE-T1WI, BBB leakage peaked at 6h and at day 3 with a transitional partial recovery around 24h. The infarct volume on T2WI, DWI and ADC map matched well with that on TTC staining at 12h and at day 1 (p>0.05). CONCLUSION: The evolution of the present photothrombotic stroke model in rats could be characterized by MRI. The obtained information may help longitudinal studies of cerebral ischemia and anti-stroke agents using the same model.

Effects of footbathing on autonomic nerve and immune function.

The purpose of this study was to investigate the effects of footbathing on autonomic nerve and immune function. Eleven healthy female volunteers (aged 22-24 years) undertook footbaths at 42 degrees C for 10 min, with or without additional mechanical stimulation (air bubbles and vibration). Autonomic responses were evaluated by electrocardiography and spectral analysis of heart rate variability, and by measurement of blood flow in the sural region. White blood cell (WBC) counts, ratios of lymphocyte subsets, and natural killer (NK) cell cytotoxicity were used as indicators of immune function. Footbathing with mechanical stimulation produced (1) significant changes in the measured autonomic responses, indicating a shift to increased parasympathetic and decreased sympathetic activity and (2) significant increases in WBC count and NK cell cytotoxicity, suggesting an improved immune status. Because these physiological changes are likely to be of benefit to health, our findings support the use of footbathing in nursing practice.

A Review of the Mechanisms of Blood-Brain Barrier Permeability by Tissue-Type Plasminogen Activator Treatment for Cerebral Ischemia.

Cerebrovascular homeostasis is maintained by the blood-brain barrier (BBB), which forms a mechanical and functional barrier between systemic circulation and the central nervous system (CNS). In patients with ischemic stroke, the recombinant tissue-type plasminogen activator (rt-PA) is used to accelerate recanalization of the occluded vessels. However, rt-PA is associated with a risk of increasing intracranial bleeding (ICB). This effect is thought to be caused by the increase in cerebrovascular permeability though various factors such as ischemic reperfusion injury and the activation of matrix metalloproteinases (MMPs), but the detailed mechanisms are unknown. It was recently found that rt-PA treatment enhances BBB permeability not by disrupting the BBB, but by activating the vascular endothelial growth factor (VEGF) system. The VEGF regulates both the dissociation of endothelial cell (EC) junctions and endothelial endocytosis, and causes a subsequent increase in vessel permeability through the VEGF receptor-2 (VEGFR-2) activation in ECs. Here, we review the possibility that rt-PA increases the penetration of toxic molecules derived from the bloodstream including rt-PA itself, without disrupting the BBB, and contributes to these detrimental processes in the cerebral parenchyma.

Dynamic susceptibility contrast-enhanced perfusion MR imaging at 1.5 T predicts final infarct size in a rat stroke model.

The purpose of the present animal experiment was to determine whether source images from dynamic susceptibility contrast-enhanced perfusion weighted imaging (DSC-PWI) at a 1.5T MR scanner, performed early after photochemically induced thrombosis (PIT) of cerebral middle artery (MCA), is feasible to predict final cerebral infarct size in a rat stroke model. Fifteen rats were subjected to PIT of proximal MCA. T2 weighted imaging (T2WI), diffusion-weighted imaging (DWI), and contrast-enhanced PWI were obtained at 1 h and 24 h after MCA occlusion. The relative lesion size (RLS) was defined as lesion volume/brain volume x 100% and measured for MR images, and compared with the final RLS on the gold standard triphenyl tetrazolium chloride (TTC) staining at 24 h. One hour after MCA occlusion, the RLS with DSC-PWI was 24.9 +/- 6.3%, which was significantly larger than 17.6 +/- 4.8% with DWI (P < 0.01). At 24 h, the final RLS on TTC was 24.3 +/- 4.8%, which was comparable to 25.1 +/- 3.5%, 24.6 +/- 3.6% and 27.9 +/- 6.8% with T2WI, DWI and DSC-PWI respectively (P > 0.05). The fact that at 1 h after MCA occlusion only the displayed perfusion deficit was similar to the final infarct size on TTC (P > 0.05) suggests that early source images from DSC-PWI at 1.5T MR scanner is feasible to noninvasively predict the final infarct size in rat models of stroke.

Role of tissue plasminogen activator/plasmin cascade in delayed neuronal death after transient forebrain ischemia.

We studied the possible involvement of the tissue plasminogen activator (t-PA)/plasmin system on both delayed neuronal death in the hippocampus and the associated enhancement of locomotor activity in rats, after transient forebrain ischemia induced by a four-vessel occlusion (FVO). Seven days after FVO, locomotor activity was abnormally increased and, after 10 days, pyramidal cells were degraded in the CA1 region of the hippocampus. FVO increased the t-PA antigen level and its activity in the hippocampus, which peaked at 4 h. Both the enhanced locomotor activity and the degradation of pyramidal cells were significantly suppressed by intracerebroventricular injection of aprotinin, a plasmin inhibitor, at 4 h but not during FVO. These results suggest the importance of the t-PA/plasmin cascade during the early pathological stages of delayed neuronal death in the hippocampus following transient forebrain ischemia.

Recombinant tissue-type plasminogen activator transiently enhances blood-brain barrier permeability during cerebral ischemia through vascular endothelial growth factor-mediated endothelial endocytosis in mice.

Recombinant tissue-type plasminogen activator (rt-PA) modulates cerebrovascular permeability and exacerbates brain injury in ischemic stroke, but its mechanisms remain unclear. We studied the involvement of vascular endothelial growth factor (VEGF)-mediated endocytosis in the increase of blood-brain barrier (BBB) permeability potentiated by rt-PA after ischemic stroke. The rt-PA treatment at 4 hours after middle cerebral artery occlusion induced a transient increase in BBB permeability after ischemic stroke in mice, which was suppressed by antagonists of either low-density lipoprotein receptor families (LDLRs) or VEGF receptor-2 (VEGFR-2). In immortalized bEnd.3 endothelial cells, rt-PA treatment upregulated VEGF expression and VEGFR-2 phosphorylation under ischemic conditions in an LDLR-dependent manner. In addition, rt-PA treatment increased endocytosis and transcellular transport in bEnd.3 monolayers under ischemic conditions, which were suppressed by the inhibition of LDLRs, VEGF, or VEGFR-2. The rt-PA treatment also increased the endocytosis of endothelial cells in the ischemic brain region after stroke in mice. These findings indicate that rt-PA increased BBB permeability via induction of VEGF, which at least partially mediates subsequent increase in endothelial endocytosis. Therefore, inhibition of VEGF induction may have beneficial effects after thrombolytic therapy with rt-PA treatment after stroke.