Danica Stanimirovic

Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and — when barrier integrity is impaired — in the pathology of many common CNS disorders such as Alzheimers disease, Parkinsons disease and stroke.

Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system

Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.

Inflammatory mediators of cerebral endothelium: a role in ischemic brain inflammation.

Brain inflammation has been implicated in the development of brain edema and secondary brain damage in ischemia and trauma. Adhesion molecules, cytokines and leukocyte chemoattractants released/presented at the site of blood‐brain barrier (BBB) play an important role in mobilizing peripheral inflammatory cells into the brain. Cerebral endothelial cells (CEC) are actively engaged in processes of microvascular stasis and leukocyte infiltration by producing a plethora of pro‐inflammatory mediators. When challenged by external stimuli including cytokines and hypoxia, CEC have been shown to release/express various products of arachidonic acid cascade with both vasoactive and pro‐inflammatory properties, including prostaglandins, leukotrienes, and platelet‐activating factor (PAF). These metabolites induce platelet and neutrophil activation and adhesion, changes in local cerebral blood flow and blood rheology, and increases in BBB permeability. Ischemic CEC have also been shown to express and release bioactive inflammatory cytokines and chemokines, including IL‐Iβ, IL‐8 and MCP‐1. Many of these mediators and ischemia in vitro and in vivo have been shown to up‐regulate the expression of both selectin and Ig‐families of adhesion molecules in CEC and to facilitate leukocyte adhesion and transmigration into the brain. Collectively, these studies demonstrate a pivotal role of CEC in initiating and regulating inflammatory responses in cerebral ischemia.

Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium

Delivery to the brain of drugs, peptides, and genes depends on the availability of brain‐specific delivery vectors. We used a phage‐displayed library of llama single‐domain antibodies (sdAbs) to enrich for species that selectively bind to and are internalized by human cerebromicrovascular endothelial cells (HCEC). Two sdAbs (FC5 and FC44) were selected, sequenced, subcloned, and expressed as fusion proteins with c‐Myc‐His5 tags. Similar to phage‐displayed sdAbs, soluble FC5 and FC44 were shown to selectively bind HCEC and to transmigrate across an in vitro human blood‐brain barrier (BBB) model. Both FC5 and FC44, in contrast to an unrelated llama sdAb, were also detected in the brain after i.v. injection into mice. These small (~14 kDa) antibodies have characteristics essential for a carrier‐vector and can be used to facilitate drug transport across the BBB.

The blood–brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood–brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor‐mediated process. FC5 taken up by human brain endothelial cells co‐localized with clathrin but not with caveolin‐1 by immunochemistry and was detected in clathrin‐enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin‐mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl‐β‐cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3‐kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, α(2,3)‐neuraminidase or Maackia amurensis agglutinin that recognizes α(2,3)‐, but not with Sambucus nigra agglutinin that recognizes α(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells‐derived lipids, suggesting that it binds luminal α(2,3)‐sialoglycoprotein receptor which triggers clathrin‐mediated endocytosis. This putative receptor may be a new target for developing brain‐targeting drug delivery vectors.

ABCG2 Is Upregulated in Alzheimer's Brain with Cerebral Amyloid Angiopathy and May Act as a Gatekeeper at the Blood–Brain Barrier for Aβ1–40 Peptides

Alzheimers disease (AD) is characterized by accumulation and deposition of Aβ peptides in the brain. Aβ deposition in cerebrovessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Since Aβ can be transported across blood–brain barrier (BBB), aberrant Aβ trafficking across BBB may contribute to Aβ accumulation in the brain and CAA development. Expression analyses of 273 BBB-related genes performed in this study showed that the drug transporter, ABCG2, was significantly upregulated in the brains of AD/CAA compared with age-matched controls. Increased ABCG2 expression was confirmed by Q-PCR, Western blot, and immunohistochemistry. Abcg2 was also increased in mouse AD models, Tg-SwDI and 3XTg. Aβ alone or in combination with hypoxia/ischemia failed to stimulate ABCG2 expression in BBB endothelial cells; however, conditioned media from Aβ-activated microglia strongly induced ABCG2 expression. ABCG2 protein in AD/CAA brains interacted and coimmunoprecipitated with Aβ. Overexpression of hABCG2 reduced drug uptake in cells; however, interaction of Aβ1–40 with ABCG2 impaired ABCG2-mediated drug efflux. The role of Abcg2 in Aβ transport at the BBB was investigated in Abcg2-null and wild-type mice after intravenous injection of Cy5.5-labeled Aβ1–40 or scrambled Aβ40–1. Optical imaging analyses of live animals and their brains showed that Abcg2-null mice accumulated significantly more Aβ in their brains than wild-type mice. The finding was confirmed by immunohistochemistry. These results suggest that ABCG2 may act as a gatekeeper at the BBB to prevent blood Aβ from entering into brain. ABCG2 upregulation may serve as a biomarker of CAA vascular pathology in AD patients.

Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT-nanoLC-MS/MS.

Cerebral ischemia rapidly initiates structural and functional changes in brain vessels, including blood‐brain barrier disruption, inflammation, and angiogenesis. Molecular events that accompany these changes were investigated in brain microvessels extracted using laser‐capture microdissection (LCM) from Sprague‐Dawley rats subjected to a 20 min transient global cerebral ischemia followed by 1, 6, or 24 h reperfusion. Proteins extracted from ∼300 LCM captured microvessels (20–100 µm) were ICAT‐labeled and analyzed by nanoLC‐MS. In‐house software was used to identify paired ICAT peaks, which were then sequenced by nanoLC‐MS/MS. Pattern analyses using k‐means clustering method classified 57 differentially expressed proteins in 7 distinct dynamic patterns. Protein function was assigned using Panther Classification system. Early reperfusion (1 h) was characterized by down‐regulation of ion pumps, nutrient transporters, and cell structure/motility proteins, and up‐regulation of transcription factors, signal transduction molecules and proteins involved in carbohydrate metabolism. The up‐regulation of inflammatory cytokines and proteins involved in the extracellular matrix remodeling and anti‐oxidative defense was observed in late reperfusion (6–24 h). The up‐regulation of IL‐1β and TGF‐1β in ischemic brain vessels was confirmed by ELISA, quantitative PCR, and/or immunohistochemistry. A biphasic postischemic (1 and 24 h) BBB opening for 3H‐sucrose was evident in the same model. Differentially expressed proteins identified in brain vessels during reperfusion are likely involved in orchestrating functional vascular responses to ischemia, including the observed BBB disruption. Haqqani A. S., Nesic M., Preston E., Baumann E., Kelly J., Stanimirovic D. Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT‐nanoLC‐MS/MS. FASEB J. 19, 1809–1821 (2005)

Interactions of EGFR and caveolin-1 in human glioblastoma cells: evidence that tyrosine phosphorylation regulates EGFR association with caveolae

Epidermal growth factor receptor (EGFR) amplification and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT–PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20–300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.

Identification of differentially expressed proteins in human glioblastoma cell lines and tumors

An in‐frame deletion of 801 bp in exons 2–7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MGΔEGFR) that confers high malignancy to tumor cells. Two‐dimensional gel electrophoresis followed by in‐gel digestion of separated spots and protein identification by LC‐MS‐MS and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MGΔEGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT‐PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low‐grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors. GLIA 42:194–208, 2003.