Liang Wang

Tissue Plasminogen Activator (tPA) Deficiency Exacerbates Cerebrovascular Fibrin Deposition and Brain Injury in a Murine Stroke Model Studies in tPA-Deficient Mice and Wild-Type Mice on a Matched Genetic Background

Although the serine protease, tissue plasminogen activator (tPA), is approved by the US Food and Drug Administration for therapy to combat focal cerebral infarction, the basic concept of thrombolytic tPA therapy for stroke was challenged by recent studies that used genetically manipulated tPA-deficient (tPA-/-) mice, which suggested that tPA mediates ischemic neuronal damage. However, those studies were potentially flawed because the genotypes of tPA-/- and wild-type control mice were not entirely clear, and ischemic neuronal injury was evaluated in isolation of tPA effects on brain thrombosis. Using mice with appropriate genetic backgrounds and a middle cerebral artery occlusion stroke model with nonsiliconized thread, which does lead to microvascular thrombus formation, in the present study we determined the risk for cerebrovascular thrombosis and neuronal injury in tPA-/- and genetically matched tPA+/+ mice subjected to transient focal ischemia. Cerebrovascular fibrin deposition and the infarction volume were increased by 8.2- and 6. 7-fold in tPA-/- versus tPA+/+ mice, respectively, and these variables were correlated with reduced cerebral blood flow up to 58% (P<0.05) and impaired motor neurological score by 70% (P<0.05). Our findings indicate that tPA deficiency exacerbates ischemia-induced cerebrovascular thrombosis and that endogenous tPA protects the brain from an ischemic insult, presumably through its thrombolytic action. In addition, our study emphasizes the importance of appropriate genetic controls in murine stroke research.

Chronic Nicotine Treatment Enhances Focal Ischemic Brain Injury and Depletes Free Pool of Brain Microvascular Tissue Plasminogen Activator in Rats

Effects of nicotine treatment (4.5 mg/kg of nicotine-free base/day administered s.c. by osmotic minipumps for 14 days) on focal ischemic stroke and expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in cerebral microvessels were studied in rats in vivo using a reversible (1 h) middle cerebral artery occlusion model. Plasma levels of nicotine and its major metabolite cotinine after 14 days of treatment were 88 and 364 ng/ml, respectively. Nicotine treatment resulted in 35–40% (p <0.001) decrease in the blood flow in the periphery of the ischemic core during reperfusion, an increase in the neurologic score of 2.6-fold (p <0.01), and 36% (p <0.05) and 121% (p <0.01) increases in the injury and edema volume in the pallium, respectively. A free pool of brain microvascular t-PA antigen was completely depleted by nicotine, while the expression of the PAI-1 antigen and/or PAI-1-t-PA complexes remained unchanged. The relative abundance of cerebromicrovascular t-PA mRNA transcript versus β-actin mRNA transcript did not change with nicotine. It is concluded that chronic nicotine treatment impairs the restoration of blood flow, worsens the neurologic outcome, and enhances brain injury following an ischemic insult. These nicotine effects are associated with depletion of brain microvascular t-PA antigen.

Glycoprotein 330/megalin (LRP-2) has low prevalence as mRNA and protein in brain microvessels and choroid plexus.

Prior studies indicated that glycoprotein 330 (gp330)/megalin mediates transcytosis of apolipoprotein J (apoJ) with Alzheimers amyloide-peptide (Abeta) across the vascular membranes of the central nervous system (CNS). Here we show the presence of gp330/megalin mRNA and gp330-like immunoepitopes in brain capillaries and choroid plexus and their absence from brain parenchyma. By polymerase chain reaction (PCR) we estimated 1.2 x 10(5) molecules (1 pg) of gp330/megalin mRNA/microg total brain capillary RNA, which is 3% of that in kidney RNA. However, gp330 mRNA was not detected by in situ hybridization in vascular CNS tissue, presumably because of low transcript prevalence. The ratio of gp330 protein:RNA was 17-fold higher in choroid plexus vs brain capillaries, which implies tissue specific regulation of the protein and mRNA prevalence. We conclude that gp330/megalin mRNA and protein are expressed in brain capillaries and choroid plexus in small amounts that are consistent with the observed activities of this endocytosing receptor in the regulation of apoJ and Abeta uptake by the CNS.

Expression of Tissue Plasminogen Activator in Cerebral Capillaries: Possible Fibrinolytic Function of the Blood-Brain Barrier

Previous work has shown that tissue plasminogen activator (tPA) is a key enzyme in the control of fibrinolysis within the vascular system. The sources of brain tPA and the mechanisms by which tPA secretion and production occur within cerebral microcirculation are not well established. In this study, expression of tPA was investigated in cerebral capillaries and capillary-depleted brain isolated from cortices of 4- to 5-week-old rats and guinea pigs. In both species, a single tPA band of M(r) 67,000 was detected in cerebral capillaries by Western blot analysis. The tPA signal was absent from capillary-depleted brain. These results were corroborated at the messenger ribonucleic acid level. Reverse transcription-polymerase chain reaction analysis revealed the presence of tPA complementary deoxyribonucleic acid in samples derived from cerebral microvessels and demonstrated very low or undetectable tPA expression in capillary-depleted brain. Immunohistochemical analysis confirmed tPA localization in endothelial cells of brain capillaries. We conclude that microvascular endothelium, i.e., the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation. Delineation of the molecular mechanisms of blood-brain barrier-mediated fibrinolysis will likely contribute to future stroke prevention efforts.

Brain Capillary Tissue Plasminogen Activator in a Diabetes Stroke Model

BACKGROUND AND PURPOSE Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury. METHODS Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion. RESULTS All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively. CONCLUSIONS Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.

Thrombomodulin Expression in Bovine Brain Capillaries Anticoagulant Function of the Blood-Brain Barrier, Regional Differences, and Regulatory Mechanisms

Thrombomodulin (TM), a key cofactor of the TM-protein C pathway, is of major biologic significance for the antithrombotic properties of endothelial cells. Yet, there is uncertainty whether TM is expressed in brain and what mechanisms govern brain endothelial anticoagulant activity. In this study, bovine brain capillaries were used as an in vitro model of the blood-brain barrier to determine factors involved in the regulation of TM expression in cerebral vasculature. Quantitative competitive-polymerase chain reaction assay revealed significant regional differences in the amount of brain capillary TM mRNA, i.e., cortical > cerebellar > pontine, consistent with the reverse transcription-polymerase chain reaction findings in which the abundance of TM mRNA was analyzed relative to beta-actin mRNA. Regional differences in TM mRNA brain capillary level correlated well with differences in protein C activation. The TM mRNA and activity were not detectable in brain parenchyma. Pathogenic mediators of ischemic stroke, interleukin 1 beta (10 U/mL), and tumor necrosis factor alpha (10 U/mL), produced a time-dependent decrease in brain capillary TM mRNA (t1/2 of 2.1 and 3.9 hours, respectively) and reduced endothelial TM activity. Incubation of brain capillaries with retinoic acid (10 mumol/L) and dibutyryl cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and 8 hours, respectively, followed by an increase in protein C activation. We conclude that TM at the blood-brain barrier is likely to be an important physiologic anticoagulant in brain microcirculation. Its downregulation by cytokines may contribute to ischemic brain damage and potentially could be counteracted by retinoic acid and cAMP.

Expression of Tissue Plasminogen Activator in Cerebral Capillaries

PREVIOUS WORK HAS shown that tissue plasminogen activator (tPA) is a key enzyme in the control of fibrinolysis within the vascular system. The sources of brain tPA and the mechanisms by which tPA secretion and production occur within cerebral microcirculation are not well established. In this study, expression of tPA was investigated in cerebral capillaries and capillary-depleted brain isolated from cortices of 4- to 5-week-old rats and guinea pigs. In both species, a single tPA band of M r 67,000 was detected in cerebral capillaries by Western blot analysis. The tPA signal was absent from capillary-depleted brain. These results were corroborated at the messenger ribonucleic acid level. Reverse transcription-polymerase chain reaction analysis revealed the presence of tPA complementary deoxyribonucleic acid in samples derived from cerebral microvessels and demonstrated very low or undetectable tPA expression in capillary-depleted brain. Immunohistochemical analysis confirmed tPA localization in endothelial cells of brain capillaries. We conclude that microvascular endothelium, i.e., the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation. Delineation of the molecular mechanisms of blood-brain barrier-mediated fibrinolysis will likely contribute to future stroke prevention efforts.

Immunohistochemical Localization of Tissue Plasminogen Activator in Vascular Endothelium of Stroke-prone Regions of the Rat Brain

OBJECTIVE Tissue plasminogen activator (tPA), a major regulator of fibrinolysis, is present in cerebrovascular endothelium. We have suggested that local regulation of tPA synthesis and release in brain microcirculation could be important determinants of the degree of damage after cerebral ischemia. In this study, the normal distribution of tPA antigen was determined in several stroke-prone regions in the rat brain often used to study the pathophysiological consequences of cerebral ischemia. METHODS Immunohistochemistry and Western blot analysis were performed using an antibody that detects free tPA antigen and tPA complexed to its rapid inhibitor, plasminogen activator inhibitor-1 (PAI-1). Staining for von Willebrand factor, a brain endothelial cell marker, served as a positive control. RESULTS Relative to von Willebrand factor, 8.6, 13, 11.4, and 20.4% of vessels in the parietal cortex, frontal cortex, striatum, and hippocampus, respectively, were tPA-positive. The majority of tPA-positive vessels (58-75%) were classified as precapillary arterioles and postcapillary venules (7-20 microm), whereas capillaries (4-7 microm) and small arterioles and venules (20-40 microm) accounted for 11 to 22% and 11 to 19%, respectively, of tPA-positive vessels. Western blot analysis of brain microvascular proteins confirmed the presence of free tPA (67 kDa) and a stronger band representing tPA-PAI-1 complexes. CONCLUSION The tPA-containing cerebrovascular endothelium is distributed mainly in smaller vessels. In addition to the free pool of tPA, a large portion of tPA is complexed to PAI-1 and is therefore functionally inactive. The size of the free tPA cerebrovascular pool may be regulated by PAI-1, which in turn could suppress fibrinolysis in the cerebral microcirculation.

Brain Injury and Cerebrovascular Fibrin Deposition Correlate with Reduced Antithrombotic Brain Capillary Functions in a Hypertensive Stroke Model

Hemostasis factors may influence the pathophysiology of stroke. The role of brain hemostasis in ischemic hypertensive brain injury is not known. We studied ischemic injury in spontaneously hypertensive rats in relation to cerebrovascular fibrin deposition and activity of different hemostasis factors in brain microcirculation. In spontaneously hypertensive rats subjected to transient middle cerebral artery occlusion versus normotensive Wistar-Kyoto (W-K) rats, infarct and edema volumes were increased by 6.1-fold (P < 0.001) and 5.8-fold (P < 0.001), respectively, the cerebral blood flow (CBF) reduced during middle cerebral artery occlusion (MCAO) by 55% (P < 0.01), motor neurologic score increased by 6.9-fold (P < 0.01), and cerebrovascular fibrin deposition increased by 6.8-fold (P < 0.01). Under basal conditions, brain capillary protein C activation and tissue plasminogen activator activity were reduced in spontaneously hypertensive rats compared with Wistar-Kyoto rats by 11.8-fold (P < 0.001) and 5.1-fold (P < 0.001), respectively, and the plasminogen activator inhibitor-1 antigen and tissue factor activity were increased by 154-fold (P < 0.00001) and 74% (P < 0.01), respectively. We suggest that hypertension reduces antithrombotic mechanisms in brain microcirculation, which may enhance cerebrovascular fibrin deposition and microvascular obstructions during transient focal cerebral ischemia, which results in greater neuronal injury.

Haemostatic Functions of the Blood-Brain Barrier: Possible Implications in the Pathogenesis of Stroke

The balance between clot-forming (procoagulant) and clot-dissolving (fibrinolytic) pathways in the brain is closely controlled, but the mechanisms responsible for its maintenance are not known. Alterations in this balance may ultimately result in cerebral infarction or stroke. Understanding haemostatic mechanisms in the brain ultimately requires the delineation of the haemostatic functions of the blood-brain barrier (BBB).