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TISSUE-SPECIFIC STEM CELLS

Adhesive Interactions Between Human Neural Stem Cells and Inflamed Human Vascular Endothelium Are Mediated by Integrins

^aBurnham Institute for Medical Research, La Jolla, California, USA;
^bLa Jolla Institute for Molecular Medicine, San Diego, California, USA

Key Words. Adhesion molecules • Rolling • Neural stem cell • Integrins • Homing • Endothelial cell • Cell trafficking

Correspondence: Evan Y. Snyder, M.D., Ph.D., Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 58-646-3158; Fax: 858-713-6273; e-mail: esnyder{at}burnham.org or Sophia K. Khaldoyanidi, M.D., Ph.D. La Jolla Institute for Molecular Medicine, 4570 Executive Drive, San Diego, California 92121, USA. Telephone: 858-587-8788; Fax: 858-587-6742; e-mail: sophia{at}ljimm.org

Received November 16, 2005; accepted for publication May 30, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussions Disclosures Acknowledgments References

 
Understanding the mechanisms by which stem cells home precisely to regions of injury or degeneration is of importance to both basic and applied regenerative medicine. Optimizing regenerative processes may depend on identifying the range of molecules that subserve stem cell trafficking. The "rolling" of extravasating cells on endothelium under conditions of physiological flow is the first essential step in the homing cascade and determines cell adhesion and transmigration. Using a laminar flow chamber to simulate physiological shear stress, we explored an aspect of this process by using human neural stem cells (hNSCs). We observed that the interactions between hNSCs and tumor necrosis factor- (TNF-)-stimulated human endothelium (simulating an inflamed milieu) are mediated by a subclass of integrins—2, 6, and ß1, but not 4, v, or the chemokine-mediated pathway CXCR4-stromal cell-derived factor-1—suggesting not only that the mechanisms mediating hNSC homing via the vasculature differ from the mechanisms mediating homing through parenchyma, but also that each step invokes a distinct pathway mediating a specialized function in the hNSC homing cascade. (TNF- stimulation also upregulates vascular cell adhesion molecule-1 expression on the hNSCs themselves and increases NSC-endothelial interactions.) The selective use of integrin subgroups to mediate homing of cells of neuroectodermal origin may also be used to ensure that cells within the systemic circulation are delivered to the pathological region of a given organ to the exclusion of other, perhaps undesired, organs.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussions Disclosures Acknowledgments References

 
Understanding the mechanisms by which "solid organ" somatic stem cells home precisely to regions of injury or degeneration (a "niche") is of importance to both basic and applied regenerative medicine. Repair by stem/progenitor cells—whether endogenous or transplanted—may be limited in part by an inability to ensure a sufficient number of reconstituting cells in the damaged area at the opportune time. Optimizing regenerative processes, therefore, may depend on first identifying the range of molecules that subserve trafficking. We explored an aspect of this process by using human neural stem cells (hNSCs) [1].

The migration of hNSCs is a tightly regulated process. Previously, we and others have shown that hNSCs rely on inflammatory chemokines for homing through parenchyma to focal areas of injury (emulated by stroke) [2]. There is also a suggestion, although controversial, that stem cells from outside the central nervous system (CNS)—either endogenous hNSCs resident in non-neural zones (e.g., bone marrow) [3, 4] or administered exogenously [5, 6]—may constitutively home to intracranial pathology. Under these situations, it is unclear what mechanism might mediate such selective homing given the characteristics of cerebrovascular endothelium, which (as a component of the blood-brain barrier) serves to separate CNS parenchyma from the vascular compartment, playing a crucial role in regulating the trafficking of cells. Such a process consists of two major phases: (a) extravasation and (b) seeding of the niche. Extravasation involves the interaction of the NSCs with the vascular endothelium under conditions of physiological flow and includes (a) the "rolling" of cells, (b) adhesion to the luminal surface of endothelial cells, and (c) transmigration across the endothelium. Thus, proper adhesive interactions between endothelial cells and the NSC are required for the successful extravasation of hNSCs. For intraparenchymal trafficking, as well, interactions between hNSCs and endothelium play a role, such as when CNS vasculature is ruptured after trauma or when less stable vessels form during the neoangiogenesis accompanying tumor formation and progression.

We report here evidence for an aspect of NSC trafficking—the critical initial step of the homing cascade (i.e., rolling)—that is regulated by their first low-affinity interaction with vascular endothelial cells and is independent of chemokine-dependent homing mechanisms. There is growing evidence for the importance of wall shear stress in the regulation of endothelial cell function [7, 8]. Shear forces induce rapid activation of signaling cascades, transcription factors, and differential gene expression in endothelial cells [9, 10]. Therefore, we used a parallel laminar flow chamber to simulate physiological shear stress conditions to investigate the mechanisms underlying cellular interactions between hNSCs and tumor necrosis factor- (TNF-)-stimulated endothelium, a condition that models many intracranial pathological processes such as traumatic injury, inflammation, and degeneration. We found that integrins 2, 6, and ß1, but not integrins 4 and v, mediate rolling of hNSCs on TNF- stimulated human vascular endothelial cells (HUVECs) in a CXCR4-stromal cell-derived factor (SDF)-1-independent manner. Of interest as well, direct TNF- stimulation of the hNSCs themselves results in upregulation of vascular cell adhesion molecule-1 (VCAM-1) expression on hNSCs and also increases hNSC-endothelial interaction.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussions Disclosures Acknowledgments References

 
hNSC Culture Conditions
A stable line of hNSCs was isolated from the ventricular zone of a late first trimester (13-week) human fetal cadaver as previously described [1, 2, 11] and expanded with mitogens without genetic manipulation or augmentation. They were maintained in neurobasal medium containing B27 supplement, Glutamax (1%; Gibco-Invitrogen, Carlsbad, CA, http://www.invitrogen.com), fibroblast growth factor-2 (20 ng/ml; EMD Biosciences, San Diego, http://www.emdbiosciences.com/html/CBC/home.html), heparin (8 µg/ml; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and leukemia inhibitory factor (10 ng/ml; Chemicon International, Temecula, CA, http://www.chemicon.com). The cells were fed twice per week. Both adherent and floating clusters were passaged. When clusters reached 10 cell diameters, they were dissociated with accutase and then split with a seeding density of 50,000 cells per cm². For fluorescence-activated cell sorting (FACS) analysis and laminar parallel flow chamber experiments, a single-cell suspension was prepared by incubating the cells in phosphate-buffered saline (PBS)-based enzyme-free cell dissociation buffer (Invitrogen). Where indicated, hNSCs were preincubated with SDF-1 (100 ng/ml; Upstate Biotechnology, Lake Placid, NY, http://www.upstatebiotech.com) or manganese (Mn, 2 mM; Sigma-Aldrich) for 5 minutes at 37°C.

In Vitro Laminar Parallel Flow Chamber Assay
The rolling and adhesion of hNSCs was assessed in vitro using a parallel-plate laminar flow chamber as previously described [12] (Fig. 1). Briefly, 22-mm² glass coverslips were coated with L-polylysine (10 µg/ml; Sigma-Aldrich) overnight at 4°C and washed twice with PBS. HUVECs (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, http://www.cambrex.com) were grown on these glass coverslips until 100% confluent in cell-type-specific media (Cambrex Bio Science Walkersville, Inc.). Defined levels of flow (wall shear stress) were applied to the coverslip in the flow chamber (100-µm thickness) by perfusing warm medium (RPMI containing 0.75 mM Ca²⁺ and Mg²⁺ and 0.2% human serum albumin) through a constant infusion syringe pump (Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com). The flow chamber was then perfused with a cellular suspension of hNSCs (10 ml at 1 x 10⁵ cells per ml) at various shear stresses (2 x 10⁵ cells per each shear stress). At least three slides with human endothelial cells were run in each experimental group. The interactions of the injected cells with the endothelial layer were observed on random fields in the central sector of each slide using an inverted phase-contrast microscope, and the images were video-recorded. Rolling hNSCs flowed slowly and demonstrated multiple discrete flow interruptions, whereas "adherent" cells remained stationary at a given point for extended periods of time (>30 seconds). (See supplemental online data for a more detailed description of the methodology employed.) The scoring was performed by trained, but blinded, researchers whose assessments, when uncoded, were in concordance in more than 98% of cases. Where indicated, hNSCs or the monolayers of human endothelial cells were preincubated with TNF- (10 ng/ml, 4 hours, 37°C); hNSCs were preincubated with function-blocking antibodies directed against integrins ß1, 1, 2, 3, 4, 5, 6, , CXCR4, and VCAM-1 (25 µg/ml, 45 minutes, room temperature; Chemicon International). SDF-1 (100 ng/ml; Upstate Biotechnology), RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1 (MCP-1; 100 ng/ml; Chemicon International), and monokine-induced by -interferon (MIG; Sigma-Aldrich) were added to the suspension of hNSCs 5 minutes prior to the experiment. All results are expressed as the number of rolling cells per field representing the average from three slides (four fields per slide).

View larger version (32K): [in this window] [in a new window]   Figure 1. "Rolling" hNSCs on "inflamed" human endothelium and the lack of involvement of the CXCR-4/SDF-1 pathway in this process. (A): Schematic of the parallel flow chamber device (upper left). hNSCs are injected through a syringe pump (to ensure a defined shear stress) into the parallel flow chamber, which contains a monolayer of endothelial cells on the bottom. That area of the flow chamber is enlarged schematically in the upper right. Injected hNSCs are noninteracting and rapidly flowing (shown in violet), or interact with low-affinity (i.e., rolling, depicted in red), or interact with high affinity (i.e., "adherent", shown in green) (The various proportions of the latter two categories are presented in supplemental online Fig. 1.) An actual representative photomicrograph, with some hNSCs appropriately colored to correspond to the "noninteracting" versus rolling versus adherent designation in the schematic, is shown in the lower right. Slow-rolling hNSCs appear as coniform cells (red) (where the point of the cone indicates the direction of flow, from the left to the right) (magnified in inset). The series of photomicrographs in the lower left demonstrates time-lapse images in which an adherent hNSC remains stationary during a representative 23-second observation period (black arrows), whereas a representative rolling hNSC (white arrows) moves slowly from left to right during the course of the same time period. (See supplemental online data for a more detailed description of the methodology employed.) (B): The rolling of hNSCs on TNF--stimulated human endothelial cells under conditions of variable shear stress was studied using the above-described parallel laminar flow chamber. The number of rolling hNSCs per field was counted, and the means of triplicates were calculated and expressed as the mean ± SD. Where indicated, human endothelium monolayers were exposed to TNF-, and hNSCs were pretreated with SDF-1 or CXCR-4-specific function-blocking antibodies. The data presented are from one experiment representative of two similar experiments. (C): ß1 integrin expression by hNSCs was determined by FACS analysis without stimulation (control) or following preincubation with SDF-1 or manganese (Mn). Isotype-matched IgG was used as the negative control. Fluorescence intensity (FL1) of samples was evaluated by FACScan. The results of one of two similar experiments are shown. (D): Expression of RGS3 and RGS16 mRNA in hNSCs. The first lane shows DNA size standards. The second and third lanes show results of one of two independent reverse transcription-polymerase chain reaction (RT-PCR) experiments in which the upper band corresponds to the correct size (approximately 320 bp) of the insert for RGS3 and RGS16. The RT-PCR product was isolated, cloned in pCRII, and sequenced. It was found to have an identical sequence for that reported for RGS3 or RGS16. RGS3 and RGS16 protein expression was also detected by fluorescence immunohistochemistry and the images captured under confocal microscopy. The cell nuclei are stained by DAPI (blue) (first column), RGS3 is shown in red and RGS16 in green (second column), and their overlay with DAPI is shown in the last column. Abbreviations: bp, base pair; DAPI, 4,6-diamidino-2-phenylindole; hNSC, human neural stem cell; IgG, immunoglobulin G; RGS, regulator of G-protein signaling; SDF-1, stromal cell-derived factor-1; Std, standard; TNF-, tumor necrosis factor-.

Gene Expression in hNSCs
Triplicate cultures of hNSCs were cultured as described above and then harvested, and total RNA was isolated using a Qiagen RNA isolation kit (Qiagen Inc., Valencia, CA, http://www1.qiagen.com). Probe preparation and chip hybridization were performed according to the manufacturer's recommendations (Illumina, San Diego, http://www.illumina.com). Full microarray analysis is provided in the supplemental online data and can be downloaded as an Excel (Microsoft Corporation, Redmond, WA, http://www.microsoft.com) spreadsheet (see online supplemental material and http://www.stemcellcommunity.org). Members of the RGS (regulator of G-protein signaling) gene family were defined as detectable if their hybridization signal intensities in all three samples were detected with at least 99% confidence.

Reverse Transcription-Polymerase Chain Reaction
Expression of the mRNA for RGS3 and RGS16 was detected by reverse transcription-polymerase chain reaction (RT-PCR). RNA was isolated from hNSCs using a kit from Qiagen Inc. A Qiagen kit was also used to make cDNA initiated by oligo(dT). A segment of the cDNA encoding regions stretching from amino acid 360–462 was detected using RT-PCR for RGS three by employing the following primers: forward primer 5'-AGA CGG CGG AAT GAG TCC CCT GG-3' and reverse primer 5'-GA GTC CAG GTT GAC CTC CTT GCA T-3'. For RGS16, the following primers were used: forward primer 5'-AAG ATC CGA TCA GCT ACC AAG C-3' and reverse primer 5'-GGG CTC GTC CAG GCT GCA GCT-3'. The derived cDNA fragments were hard copied into pCR II (Invitrogen) and sequenced using automated DNA sequencing employing a capillary ABI 3730 sequencer (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com).

Immunohistochemistry
A single-cell suspension of hNSCs was seeded on fibronectin-coated glass slides (10 mg/ml, 1 hour at 37°C), fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X100, and used for the intracellular detection of RGS3 and RGS16 proteins. Rabbit anti-human RGS3-specific antibody (1:500; Novus Biologicals, Inc., Littleton, CO, http://www.novusbio.com) and chicken anti-human RGS16-specific antibody (1:500; Chemicon International) staining was visualized with goat anti-rabbit Alexa-Flour 594-conjugated (Invitrogen) and goat anti-chicken Alexa-Fluor 488-conjugated (Aves Labs, Inc., Tigard, OR, http://www.aveslab.com) secondary antibodies (1:1,000). Images were captured with an Olympus FLUOVIEW FV 1,000 (x60 objective) (Olympus, Tokyo, http://www.olympus-global.com) and analyzed using Open Lab software (Improvision Inc., Lexington, MA, http://www.improvision.com).

	RESULTS AND DISCUSSIONS

Top Abstract Introduction Materials and Methods Results and Discussions Disclosures Acknowledgments References

 
hNSCs were isolated and expanded (with mitogens alone) from the primary CNS germinal zone (ventricular zone) of a fetal forebrain as previously described [1, 2] and maintained as a stable line to ensure reproducible conditions across experiments. These hNSCs are known to home in vivo to intracranial pathologies such as stroke lesions and tumors [1, 2, 11] as well as to participate in normal cerebrogenesis (including primate) [2, 13]. For these studies, we used a parallel laminar flow chamber assay to investigate the mechanisms mediating the "homing cascade"-initiating adhesive interactions (called rolling) between hNSCs and inflamed human endothelium (Fig. 1A). (See supplemental online data for a more detailed description of the methodology employed.) We allowed hNSCs to interact with untreated or TNF--exposed human endothelial cell monolayers by infusing them into the laminar flow chamber. TNF- stimulation of human endothelium emulates their condition in a range of intracranial pathological conditions, particularly those accompanied by an inflammatory signature. The number of hNSCs rolling on TNF--exposed human endothelium was significantly higher compared with control (Fig. 1B). As noted above, rolling is the initial step in somatic cell engagement with endothelium. The term refers to those cells that are impeded from flowing briskly through the chamber by instead making intermittent low-affinity contacts with the endothelium along its length, creating the appearance of rolling along its surface (Fig. 1A, schematic). In the blood stream, circulating cells undergo high shear stress; rolling is the mechanism that selectively slows down subsets of cells (out of larger population of all fast-flowing cells) to permit their ultimate firm adhesion and transmigration. This process is essential preparation for cell adhesion; blocking rolling, blocks adhesion and ultimately homing [14]. The molecular mechanisms that mediate rolling—a process that adds an essential dimension of selectivity—are distinct from those leading to adhesion [15, 16] (relative proportions for which are presented in supplemental online Fig. 1A). Those involved in NSC rolling have yet to be investigated.

Because TNF- upregulates the expression of VCAM-1 on human endothelium [17–19] (supplemental online Fig. 2) and because hNSCs express the chemokine receptor CXCR4 [2] (Fig. 2A), we anticipated that the interaction between hNSCs and TNF--exposed endothelium would be facilitated by SDF-1 (the cognate ligand for CXCR4). Interestingly, the number of rolling hNSCs was unchanged in the presence of SDF-1 (Fig. 1B). Furthermore, the number of hNSCs that rolled on TNF--treated human endothelium was not significantly inhibited by CXCR-4-specific antibodies, as compared with control isotype-matched IgG (Fig. 1B). In accordance with this observation, FACS analysis showed that activation of integrin ß1, which is normally stimulated by SDF-1 stimulation [20], was not influenced by SDF-1 in these cells (Fig. 1C). Therefore, the SDF-1/CXCR4 pathway did not appear to predominate in these cells under these conditions. The function of CXCR4 can be negatively regulated by a family of proteins named "regulator of G-protein signaling" (RGS). Previously, CXCR4 expression without a response to SDF-1 has been observed in lymphocytes that express RGS3 and RGS16 [21–24]. We asked whether a similar mechanism might be operative in the nervous system as well (i.e., whether these molecules are expressed by hNSCs). Gene expression profiling of hNSCs demonstrated the expression of 12 members of the RGS family (RGS 3, 4, 5, 6, 7, 11, 12, 13, 14, 16, 17, and 20). RGS3 and RGS16 are of particular interest because they have been shown to inhibit CXCR4 signaling. Their expression at the mRNA and protein levels was confirmed in the hNSC (Fig. 1D), suggesting that RGS expression might determine whether the CXCR-4/SDF-1 pathway functions in these cells.

View larger version (29K): [in this window] [in a new window]   Figure 2. Role of integrins in hNSC-inflamed human endothelial cell interactions under conditions of shear stress. (A): The expression of 1, 2, 3, 4, 5, 6, , and CXCR-4 on hNSCs was determined by FACS analysis. Isotype-matched IgG was used as the negative control. Fluorescent intensity (FL1) of samples was evaluated by FACScan. The results of one out of two similar experiments are shown. (B, C): Rolling of hNSCs on inflamed human endothelium under conditions of variable shear stress was studied using a parallel laminar flow chamber. (B): Few rolling hNSCs were observed under control conditions, whereas a significantly greater number were apparent after TNF- stimulation of human endothelium. Rolling was inhibited (returned to essentially control levels) when the hNSCs were preincubated with anti-2, anti-6, and anti-ß1 antibodies. The number of rolling hNSCs per field was counted; the means of triplicates are expressed as the mean ± SD. The data presented are from one experiment representative of six similar experiments. (C): Quantification of the failure of anti-integrin 4 and v antibodies to inhibit hNSC rolling. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; hNSC, human neural stem cell; IgG, immunoglobulin G; RGS, regulator of G-protein signaling; TNF-, tumor necrosis factor-.

Given that chemokine-mediated pathways appeared not to be operative in the regulation of hNSC rolling on inflamed human endothelium, we sought to determine which molecules might be pivotal. FACS analysis demonstrated that, in addition to ß1, hNSCs express high levels of integrins 2, 6, and on their surface. No or low expression of integrins 1, 3, 4, and 5 was detected (Fig. 2A). To address the question of whether these integrins mediate low-affinity adhesive interactions of hNSC with TNF--stimulated human endothelium under conditions of physiological shear stress, function-blocking antibodies directed against specific integrins were tested in the parallel laminar flow chamber assay. Antibodies directed against 2, 6, and ß1 inhibited rolling of hNSCs on TNF--stimulated human endothelial cells (Fig. 2B), whereas those against 4 and v had no significant effect (Fig. 2C). Taken together, these data suggest that integrins 2, 6, and ß1 (but not 4 and ) mediate the initial interaction (i.e., rolling) between hNSCs and injured human endothelium.

Up to this point, our study entailed examining the effect of an inflamed (i.e., TNF--stimulated) human endothelium on hNSCs. Because, in an actual inflammatory niche in vivo, the hNSCs are also exposed to inflammation, we next examined whether TNF- stimulation of the hNSCs themselves might impair their ability to interact with the vasculature. Therefore, we first determined whether TNF- changed the expression of 4 integrin and VCAM-1 on hNSCs. Whereas 4 integrin expression remained unchanged (i.e., low), TNF- actually increased the expression of VCAM-1 (Fig. 3A). Furthermore, TNF--treated hNSCs evinced increased rolling under shear stress conditions, which could be blocked by a VCAM-1-specific antibody but not by an 4-specific antibody (Fig. 3B).

View larger version (25K): [in this window] [in a new window]   Figure 3. Effect of TNF--stimulation of hNSCs on its interaction with human endothelial cells under conditions of shear stress. (A): The expression of 4 integrin and VCAM-1 on TNF--treated hNSCs was determined by FACS analysis. Isotype-matched IgG was used as the negative control. Fluorescent intensity (FL1) of samples was evaluated by FACScan. The results of one out of two similar experiments are shown. (B): The increased rolling of TNF--treated hNSCs on human endothelium was inhibited by VCAM-1-specific antibodies, but not by 4-specific antibodies. Abbreviations: FACS, fluorescence-activated cell sorting; hNSC, human neural stem cell; NT, non-treated; TNF-, tumor necrosis factor-; VCAM-1, vascular cell adhesion molecule-1.

	DISCLOSURES

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results and Discussions Disclosures Acknowledgments References

 
This work was supported by National Institutes of Health grants R21DK067084 and K18 HL081096 (S.K.K.), Studienstiftung des Deutschen Volkes (F-J.M.), University of California Tobacco-Related Disease Research Program Postdoctoral Fellowship 14FT-0126 (N.S.), and Franklin Delano Roosevelt Scholars Award from the March of Dimes (E.Y.S.). We thank Sirak Simavoryan for his help with flow chamber experiments. F-J.M. and N.S. contributed equally to this work. F.-J.M. is currently affiliated with Zentrum für Integrative Psychiatrie, 24105 Kiel, Germany.

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