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Biomedical subjects

B V Zlokovic

Publications and source records attributed to B V Zlokovic.

At least 19 recordsLinked to original sources
PubMed2001

Endothelin-1 and monocyte chemoattractant protein-1 modulation in ischemia and human brain-derived endothelial cell cultures.

Brain tissue damage due to ischemia/reperfusion has been shown to be caused, in part, by activated macrophages infiltrating into the post-ischemic brain. Using the Middle Cerebral Artery Occlusion (MCAO) mouse model, this study demonstrated that, in vivo, both endothelin-1 (Et-1), a potent vasoconstrictor, and the macrophage chemokine, monocyte chemoattractant factor-1 (MCP-1) are induced in ischemia. Further studies, using human brain-derived endothelial cells (CNS-EC), showed that in vitro, Et-1 can directly stimulate MCP-1 mRNA expression and MCP-1 protein; and this Et-1-induced MCP-1 production is mediated by the ET(A) receptor. Inflammatory cytokines, tumor necrosis factor alpha and interleukin-1beta, functioned additively and synergistically, respectively, with Et-1 to increase this MCP-1 production. Partial elucidation of the signal transduction pathways involved in Et-1-induced MCP-1 production demonstrated that protein kinase C-, but not cAMP-dependent pathways are involved. These data demonstrate that Et-1, functioning as an inflammatory peptide, increased levels of MCP-1, suggesting a mechanism for chemokine regulation during ischemia/reperfusion injury.

Animals↗
PubMed2001

Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke.

BACKGROUND: Activated protein C (APC) contributes to systemic anticoagulant and anti-inflammatory activities. APC may reduce organ damage by inhibiting thrombin generation and leukocyte activation. Neutrophils and cerebrovascular thrombosis contribute to ischemic neuronal injury, suggesting that APC may be a potential protective agent for stroke. METHODS AND RESULTS: We examined the effects of APC in a murine model of focal ischemia. After middle cerebral artery occlusion/reperfusion, the average survival time in controls was 13.6 hours. Animals that received purified human plasma-derived APC 2 mg/kg IV either 15 minutes before or 10 minutes after stroke induction survived 24 hours and were killed for neuropathological analysis. APC 2 mg/kg given before or after onset of ischemia restored cerebral blood flow, reduced brain infarct volume (59% to 69%; P:<0.003) and brain edema (50% to 61%; P:<0.05), eliminated brain infiltration with neutrophils, and reduced the number of fibrin-positive cerebral vessels by 57% (P:<0.05) and 25% (nonsignificant), respectively. The neuroprotective effect of APC was dose-dependent and associated with significant inhibition of ICAM-1 expression on ischemic cerebral blood vessels (eg, 61% inhibition with 2 mg/kg APC). Intracerebral bleeding was not observed with APC. CONCLUSIONS: APC exerts anti-inflammatory, antithrombotic, and neuroprotective effects in stroke. Central effects of APC are likely to be related to improved maintenance of the blood-brain barrier to neutrophils and to reduced microvascular obstructions and fibrin deposition.

Animals↗
PubMed2001

Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist.

OBJECTIVE: Brain tumors are highly angiogenic, and their growth and spread depend on the generation of new blood vessels. We examined the effect of the cyclic peptide antagonist pentapeptide EMD 121974, an antiangiogenic agent, on orthotopic and heterotopic brain tumor growth. METHODS: The human brain tumor cell lines DAOY (medulloblastoma) and U87 MG (glioblastoma) were injected into either the forebrain (orthotopic) or the subcutis (heterotopic) of nude mice, and daily systemic treatment with the active peptide was initiated after tumors were established. RESULTS: All control animals with orthotopic brain tumors and that received the inactive peptide EMD 135981 daily died as a result of tumor progression within 4 to 6 weeks; tumors measured 3 to 5 mm in diameter. In contrast, mice with orthotopic tumors that were treated daily with the active peptide survived for more than 16 weeks, and histological examination of the brains after 4, 8, and 12 weeks showed either no tumors or microscopic residual tumors. The growth of these brain tumor cells injected simultaneously or separately into the subcutis of nude mice (heterotopic model) was not affected by the active peptide, suggesting that the brain environment is a critical determinant of brain tumor susceptibility to growth inhibition by this pentapeptide. CONCLUSION: The cyclic pentapeptide EMD 121974 may become a treatment option specific to brain tumors. Because of its antiangiogenic effect, its use may be especially indicated after tumors are removed surgically.

Allantois↗
PubMed2000

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.

Animals↗
PubMed2000

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier.

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2) </= 25 minutes), mainly by vascular transport across the blood-brain barrier (BBB). The efflux transport system for Ass(1-40) at the BBB was half saturated at 15.3 nM, and the maximal transport capacity was reached between 70 nM and 100 nM. Ass(1-40) clearance was substantially inhibited by the receptor-associated protein, and by antibodies against LDL receptor-related protein-1 (LRP-1) and alpha(2)-macroglobulin (alpha(2)M). As compared to adult wild-type mice, clearance was significantly reduced in young and old apolipoprotein E (apoE) knockout mice, and in old wild-type mice. There was no evidence that Ass was metabolized in brain interstitial fluid and degraded to smaller peptide fragments and amino acids before its transport across the BBB into the circulation. LRP-1, although abundant in brain microvessels in young mice, was downregulated in older animals, and this downregulation correlated with regional Ass accumulation in brains of Alzheimer's disease (AD) patients. We conclude that the BBB removes Ass from the brain largely via age-dependent, LRP-1-mediated transport that is influenced by alpha(2)M and/or apoE, and may be impaired in AD.

Aging↗
PubMed2000

Differential regulation of leptin transport by the choroid plexus and blood-brain barrier and high affinity transport systems for entry into hypothalamus and across the blood-cerebrospinal fluid barrier.

Leptin is a circulating hormone that controls food intake and energy homeostasis. Little is known about leptin entry into the central nervous system (CNS). The blood-cerebrospinal fluid (CSF) barrier at the choroid plexus and the blood-brain barrier (BBB) at the cerebral endothelium are two major controlling sites for entry of circulating proteins into the brain. In the present study, we characterized leptin transport across the blood-CSF barrier and the BBB by using a brain perfusion model in lean rats. Rapid, high-affinity transport systems mediated leptin uptake by the hypothalamus (KM = 0.2 ng/ml) and across the blood-CSF barrier (KM = 1.1 ng/ml). High affinity in vivo binding of leptin was also detected in the choroid plexus (KD = 2.6 ng/ml). In contrast, low affinity carriers for leptin (KM = 88 to 345 ng/ml) were found at the BBB in the CNS regions outside the hypothalamus (e.g. cerebral cortex, caudate nucleus, hippocampus). Our findings suggest a key role of high affinity leptin transporters in the hypothalamus and choroid plexus in regulating leptin entry into the CNS and CSF under physiological conditions. Low affinity transporters at the BBB outside the hypothalamus could potentially contribute to overall neuropharmacological effects of exogenous leptin.

Animals↗
PubMed1999

Exogenous microglia enter the brain and migrate into ischaemic hippocampal lesions.

We compared migration of systemically injected microglia into normal brain vs. ischaemic brain using a model of ischaemic hippocampal lesion. Microglia were labeled by a fluorescent dye using our standard phagocytosis procedure of microscopic particles and then injected intra-arterially into Mongolian gerbils subjected to ischaemia reperfusion neuronal injury. Delayed death of pyramidal neurons was confirmed by conventional histological analysis and dUTP nick end labeling (TUNEL) method. Clusters of dye-tagged cells migrating into the hippocampal ischaemic lesions were confirmed histochemically to be microglia. Since peripherally injected microglia exhibit specific affinity for ischaemic brain lesions and does not exacerbate ischaemic neuronal injury in the present model, we suggest that microglia may have a potential to be used as a piggy-back ride to deliver therapeutic genes and/or drugs for CNS repair following transitory global ischaemic insult.

Animals↗
PubMed1999

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 Alzheimer's 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.

Animals↗
PubMed1999

Cereport (RMP-7) increases the permeability of human brain microvascular endothelial cell monolayers.

PURPOSE: To study Cereport (RMP-7, bradykinin B2 agonist) effects on human brain microvascular endothelial cell (HBMEC) monolayer permeability. METHODS: HBMEC grown on transwell membranes were exposed to Cereport. The monolayer permeability was determined with [14C]-inulin (MW. 5,200) and [3H]-dextran (MW. 70,000). RESULTS: Cereport increased the HBMEC permeability to [14C]-inulin, but not to [3H]-dextran. The effect was transient, maximal at 15 min (i.e., 79.3% increase), and polarized to the basolateral membrane. An inverted U, dose-response curve was observed with active concentrations of Cereport from 0.01 to 0.5 nmol/L, the plateau maximal effect between 0.5 and 10 nmol/L, and loss of activity at the highest concentration, i.e., 20 nmol/L. Cyclic AMP-specific phosphodiesterase 3 (PDE3) inhibitor rolipram (10 micromol/L) abolished Cereport effects, while cGMP-specific PDE5 inhibitor, zaniprast (50 micromol/L) enhanced by 31% (p < 0.05) the effect of 0.1 nmolL Cereport. Unlabeled Cereport displaced [125I]-bradykinin and/or [125I]-Cereport from the basolateral side. There was no specific Cereport binding to the apical side. CONCLUSIONS: Cereport exerts specific time, dose and size dependent actions on HMBEC monolayer that are restricted to the basolateral membrane. Its effects can be further modulated through changes in cAMP and cGMP second messenger systems.

Binding, Competitive↗
PubMed1999

Corneal transport of circulating glutathione in normal and galactosemic guinea pigs.

PURPOSE: To study synthesis and transport of glutathione (GSH) in guinea-pig cornea and to determine the effect of galactose feeding and growth on transport. METHODS: Steady-state level and maximal rate of synthesis of GSH (GSH-SR) were determined in guinea pigs fed 50% galactose diet for 10 days and in controls. By using a model of in situ eye perfusion, corneal transport of [35S]GSH (4 nM) and [14C]sucrose was measured as a function of time (1-10 min) in normal guinea pigs under gamma-glutamyltranspeptidase (GGT)-inhibited and uninhibited conditions. The unidirectional constant of GSH uptake was determined as a function of GSH concentration in the perfusate. The effect of galactose feeding on corneal uptake of tracer GSH was determined in control and 10-day galactose-fed guinea pigs. Levels of GSH and uptake of GSH also were measured in corneas from three different age groups: 10 days, 4 weeks, and 9 months. RESULTS: The mean GSH level (nmol/ mg protein) in corneas of control guinea pigs was 47.6, which decreased to 36.4 (p<0.05) in 10-day galactose-fed animals. The GSH-SR was not significantly different in the two groups (1.98 vs. 2.27 nmol/min/mg protein, respectively; p = NS). Corneal uptake of tracer [35S]GSH (4 nM) was linear up to 10 min and was several-fold higher than that of the impermeable marker [14C]sucrose. The unidirectional rate constant (corrected for sucrose) for GSH was 4.03+/-0.21x10(-3)/min. GSH uptake in normal guinea pigs occurred by a saturable process with a Km of 24+/-4 microM and Vmax of 92+/-14 pmol/min/g and was significantly inhibited by GSH and GSH monoethyl ester at 60 microM concentrations. Corneal uptake of tracer GSH in galactose-fed guinea pigs showed a dramatic decrease (almost to that of sucrose) as compared with control guinea pigs. GSH uptake was similar in corneas of 10-day and 4-week-old guinea pigs, whereas that in 9-month-old guinea pigs showed a significant (approximately 80%) decrease in uptake. CONCLUSION: Cellular uptake of GSH by the cornea in the young, adult guinea pigs is carrier mediated via mechanism(s) that can be dissociated from the transpeptidation pathway. The reduced availability of circulating GSH may be an important factor in the development of corneal pathology associated with aging and corneal hydration due to relative GSH deficiency.

Aging↗
PubMed1999

Retroviral vector-mediated transfer and expression of human tissue plasminogen activator cDNA in bovine brain endothelial cells.

OBJECTIVES: Gene transfer of thrombolytic enzymes to vascular endothelial cells may influence the kinetics of intravascular thrombosis. This study defines the potential for gene transfer of tissue plasminogen activator (tPA) into bovine brain endothelial cells (BBEC). METHODS: The retroviral vectors derived from murine leukemia virus (MuLV) were used to transfer human tPA cDNA to BBEC. The tPA activity, tPA antigen and tPA inhibitor 1 (PAI-1) antigen were determined in the supernatant of transduced (BBEC/tPA) cell cultures by an immunoassay. RESULTS: The tPA antigen and enzymatic activity in cell culture supernatants of BBEC/tPA transduced cells were 75 ng/ml and 14 IU/ml after 4 days, that was 25 and 28-fold higher compared to the respective values in control cells. The PAI-1 antigen was not affected by tPA cDNA transfer. The Western blot assay of cell lysates confirmed that the majority of tPA in BBEC/tPA transduced cells was in the form of free tPA. While the maximal transduction efficiency of BBEC with an amphotropic MuLV vector was about 15%, a MuLV pseudotyped with vesicular stomatitis virus G glycoprotein envelope achieved high > 90% maximal transduction efficiency. CONCLUSIONS: The fibrinolytic activity of brain endothelial cells can be enhanced by transferring human tPA cDNA. These findings provide an initial step in implementation of future studies that investigate the use of this technology as an adjunctive treatment for cerebrovascular disease.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

Rat brain capillary thrombomodulin: structure and function.

The anticoagulant transmembrane glycoprotein thrombomodulin (TM) is expressed at the luminal surface of vascular endothelial cells. Recently, we showed that TM antigen and TM mRNA are expressed in brain microvessels in several species and that brain capillaries have the capability to activate protein C. The activation of protein C in brain microcirculation was greatly impaired by major stroke risk factors in rats due to downregulation of TM. In this study, a partial sequence of TM was determined from TM mRNA from brain capillaries examined in brain capillaries of the rat, a species that provides a useful model to investigate stroke mechanisms in relation to brain hemostasis. The predicted deduced amino acid sequences for rat TM were compared with other TM sequences. Particularly high homology (77-100%) among functional domains of the protein, i.e., the epidermal growth factor repeats (EGFRs) 1-6 and the transmembrane region, was observed between mice and rats. Somewhat less degree of homology was observed for bovine and human EGFRs 1-6, while the homology of the transmembrane region was 92-96%. All cysteine residues were conserved among the TM sequences, and specific amino acids previously suggested to be essential for activation of protein C by thrombin TM were highly conserved. We conclude that the highly conserved mRNA and protein sequences may reflect a similar anticoagulant role of TM in brain endothelial and systemic vascular endothelial cells across different species.

Amino Acid Sequence↗
PubMed1998

Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis.

Alzheimer Disease↗
PubMed1998

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy.

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.

3T3 Cells↗
PubMed1998

Cerebrovascular accumulation and increased blood-brain barrier permeability to circulating Alzheimer's amyloid beta peptide in aged squirrel monkey with cerebral amyloid angiopathy.

Senescent squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Cerebrovascular sequestration and blood-brain barrier (BBB) permeability to 121I-amyloid beta(1-40) synthetic peptide (sA beta(1-40)) were studied in adult versus aged squirrel monkey 1 h after a single intravenous injection. In aged monkey, the half-time of elimination of sA beta(1-40), t(1/2)e, was prolonged by 0.6 h, the systemic clearance, ClSS, was reduced from 1.8 to 1.1 ml/min/kg, and the mean residence time of intact peptide in the circulation was increased by 1 h (45%). In adult monkey, cerebrovascular sequestration of intact sA beta(1-40) was significant, and the BBB permeability was 18.6-fold higher than for inulin. In aged monkey, the sequestration of intact sA beta(1-40) by cortical and leptomeningeal microvessels and the BBB permeability were increased by 5.9, 1.8-, and 2.1-fold, respectively, in the presence of an unchanged barrier to inulin. In brain parenchyma of aged animals, 76.1% of circulating sA beta(1-40) remained intact versus 45.7% in adult. We conclude that multiple age-related systemic effects, i.e., reduced body elimination and systemic clearance of sA beta(1-40), and reduced peripheral metabolism, may act in concert with BBB mechanisms, i.e., increased transendothelial transport and microvascular accumulation of blood-borne sA beta(1-40), and reduced brain metabolism to enhance the development of CAA.

Aging↗