Ewa Baumann

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)

Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering: EFFECTS OF INHIBITORS*

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.

A novel platform for engineering blood-brain barrier-crossing bispecific biologics

The blood‐brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic “arm” is combined with a BBB‐transcytosing arm can significantly enhance their brain delivery. The BBB‐permeable single‐domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single‐domain antibody phage display library. In this study, FC5 was engineered as a mono‐ and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30‐fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5‐compared with control domain antibody‐Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB‐impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5‐Fc fusion proteins. Improved serum pharmacokinetics of Fc‐fused FC5 contributed to a 60‐fold increase in pharmacological potency compared with the single‐domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB‐carrier arm in bispecific antibodies or antibody‐drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.—Farrington, G. K., Caram‐Salas, N., Haqqani, A. S., Brunette, E., Eldredge, J., Pepinsky, B., Antognetti, G., Baumann, E., Ding, W., Garber, E., Jiang, S., Delaney, C., Boileau, E., Sisk, W. P., Stanimirovic, D. B., A novel platform for engineering blood‐brain barrier‐crossing bispecific biologics. FASEB J. 28, 4764–4778 (2014). www.fasebj.org

Post-ischemic hypothermia attenuates loss of the vascular basement membrane proteins, agrin and SPARC, and the blood–brain barrier disruption after global cerebral ischemia

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague-Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42-45 mm Hg). In a separate group of animals, a mild (32 degrees C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from approximately 500 brain vessels (20-100 microm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using (3)H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p<0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCM-captured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h post-ischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for (3)H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.

Dynamic Analysis of the Blood-Brain Barrier Disruption in Experimental Stroke Using Time Domain In Vivo Fluorescence Imaging

The blood-brain barrier (BBB) disruption following cerebral ischemia can be exploited to deliver imaging agents and therapeutics into the brain. The aim of this study was (a) to establish novel in vivo optical imaging methods for longitudinal assessment of the BBB disruption and (b) to assess size selectivity and temporal patterns of the BBB disruption after a transient focal ischemia. The BBB permeability was assessed using in vivo time domain near-infrared optical imaging after contrast enhancement with either free Cy5.5 (1 kDa) or Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa) in mice subjected to either 60- or 20-minute transient middle cerebral artery occlusion (MCAO) and various times of reperfusion (up to 14 days). In vivo imaging observations were corroborated by ex vivo brain imaging and microscopic analyses of fluorescent tracer extravasation. The in vivo optical contrast enhancement with Cy5.5 was spatially larger than that observed with BSA-Cy5.5. Longitudinal studies after a transient 20-minute MCAO suggested a bilateral BBB disruption, more pronounced in the ipsilateral hemisphere, peaking at day 7 and resolving at day 14 after ischemia. The area differential between the BBB disruption for small and large molecules could potentially be useful as a surrogate imaging marker for assessing perinfarct tissues to which neuroprotective therapies of appropriate sizes could be delivered.

The epithelial membrane protein 1 is a novel tight junction protein of the blood-brain barrier.

In the central nervous system, a constant microenvironment required for neuronal cell activity is maintained by the blood—brain barrier (BBB). The BBB is formed by the brain microvascular endothelial cells (BMEC), which are sealed by tight junctions (TJ). To identify genes that are differentially expressed in BMEC compared with peripheral endothelial cells, we constructed a subtractive cDNA library from porcine BMEC (pBMEC) and aortic endothelial cells (AOEC). Screening the library for differentially expressed genes yielded 26 BMEC-specific transcripts, such as solute carrier family 35 member F2 (SLC35F2), ADP-ribosylation factor-like 5B (ARL5B), TSC22 domain family member 1 (TSC22D1), integral membrane protein 2A (ITM2A), and epithelial membrane protein 1 (EMP1). In this study, we show that EMP1 transcript is enriched in pBMEC compared with brain tissue and that EMP1 protein colocalizes with the TJ protein occludin in mouse BMEC by coimmunoprecipitation and in rat brain vessels by immunohistochemistry. Epithelial membrane protein 1 expression was transiently induced in laser-capture microdissected rat brain vessels after a 20-min global cerebral ischemia, in parallel with the loss of occludin immunoreactivity. The study identifies EMP1 as a novel TJ-associated protein of the BBB and suggests its potential role in the regulation of the BBB function in cerebral ischemia.

In vivo time domain optical imaging of renal ischemia-reperfusion injury: discrimination based on fluorescence lifetime.

Fluorescence lifetime is an intrinsic parameter of the fluorescent probe, independent of the probe concentration but sensitive to changes in the surrounding microenvironment. Therefore, fluorescence lifetime imaging could potentially be applied to in vivo diagnostic assessment of changes in the tissue microenvironment caused by disease, such as ischemia. The aim of this study was to evaluate the utility of noninvasive fluorescence lifetime imaging in distinguishing between normal and ischemic kidney tissue in vivo. Mice were subjected to 60-minute unilateral kidney ischemia followed by 6-hour reperfusion. Animals were then injected with the near-infrared fluorescence probe Cy5.5 or saline and imaged using a time-domain small-animal optical imaging system. Both fluorescence intensity and lifetime were acquired. The fluorescence intensity of Cy5.5 was clearly reduced in the ischemic compared with the contralateral kidney, and the fluorescence lifetime of Cy5.5 was not detected in the ischemic kidney, suggesting reduced kidney clearance. Interestingly, the two-component lifetime analysis of endogenous fluorescence at 700 nm distinguished renal ischemia in vivo without the need for Cy5.5 injection for contrast enhancement. The average fluorescence lifetime of endogenous tissue fluorophores was a sensitive indicator of kidney ischemia ex vivo. The study suggests that fluorescence lifetime analysis of endogenous tissue fluorophores could be used to discriminate ischemic or necrotic tissues by noninvasive in vivo or ex vivo organ imaging.

Multiplexed Evaluation of Serum and CSF Pharmacokinetics of Brain- Targeting Single-Domain Antibodies Using a NanoLC−SRM-ILIS Method

FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.1. FC5 and FC44 compared to control VHHs demonstrated significantly (p < 0.01) enhanced transport (50-100-fold) across rat in vitro BBB model as well as in vivo brain targeting assessed by optical imaging. The multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed VHHs demonstrated that while all 4 VHHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB.

In Vivo Optical Imaging of Ischemic Blood–Brain Barrier Disruption

The blood-brain barrier (BBB) disruption following cerebral ischemia (stroke) contributes to the development of life-threatening brain edema. Recent studies suggested that the ischemic BBB disruption is not uniform throughout the affected brain region. The aim of this study was to establish in vivo optical imaging methods to assess the size selectivity and spatial distribution of the BBB disruption after a focal cerebral ischemia. The BBB permeability was assessed in mice subjected to a 60-min middle cerebral artery occlusion and 24 h of reperfusion using in vivo time domain near-infrared optical imaging after contrast enhancement with two tracers of different molecular size, Cy5.5 (1 kDa) and Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa). Volumetric reconstruction of contrast-enhanced brain areas in vivo and ex vivo indicated that the BSA-Cy5.5-enhancement is identical to the volume of infarct determined by TTC staining, whereas the volume of enhancement with Cy5.5 was 40% greater. The volume differential between areas of BBB disruption for small and large-size molecules could be useful for determining the size of peri-infarct tissues (penumbra) that can respond to neuroprotective therapies.

Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier:

Current methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.