Matthias U. Nollert

Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions.

Both P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin are localized on the microvilli of neutrophils and have been implicated in the attachment of neutrophils to P-selectin or E-selectin. We directly compared the attachment and rolling of neutrophils on P-selectin and E-selectin under flow, with emphasis on the functions of PSGL-1 and L-selectin. Flowing neutrophils attached more avidly and rolled at lower velocities on P-selectin than on E-selectin at matched densities. Studies with purified molecules indicated that P-selectin and E-selectin bound to a related site on PSGL-1 that overlapped the epitope for the anti-PSGL-1 mAb PL1. E-selectin bound with lower affinity than P-selectin to this site and also bound to an additional site(s) on PSGL-1.PL1 abolished adhesion of neutrophils to P-selectin under shear or static conditions, whereas DREG-56, a mAb to L-selectin, had no effect on adhesion to P-selectin. PL1 inhibited attachment of neutrophils to E-selectin under flow but not static conditions. DREG-56 also inhibited attachment of flowing neutrophils to E-selectin, and a combination of DREG-56 and PL1 nearly eliminated attachment to E-selectin under flow. These data suggest that PSGL-1 functions cooperatively with L-selectin to mediate optimal attachment of flowing neutrophils to E-selectin but not to P-selectin. Neutrophils attach more efficiently and with greater strength to P-selectin, perhaps because of the higher affinity of P-selectin for the PL1-defined site on PSGL-1.

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow.

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin–binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-β-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin–ligand interactions.

Dimerization of a selectin and its ligand stabilizes cell rolling and enhances tether strength in shear flow.

Selectins mediate rolling of leukocytes by rapid formation and dissociation of selectin–ligand bonds, which are assumed to require high mechanical strength to prevent premature dissociation by the forces applied in shear flow. This assumption is based largely on the observation that increasing wall shear stress increases only modestly the dissociation of transient leukocyte tethers on very low selectin densities. P-selectin binds to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. Both PSGL-1 and P-selectin are extended homodimers. We perfused transfected cells expressing wild-type dimeric PSGL-1 or a chimeric monomeric form of PSGL-1 on immobilized dimeric or monomeric forms of P-selectin. Cells expressing dimeric or monomeric PSGL-1 tethered to P-selectin at equivalent rates. However, cells expressing dimeric PSGL-1 established more stable rolling adhesions, which were more shear resistant and exhibited less fluctuation in rolling velocities. On low densities of dimeric P-selectin, increasing wall shear stress more rapidly increased transient tether dissociation of cells expressing monomeric PSGL-1 than dimeric PSGL-1. Tether dissociation on low densities of monomeric P-selectin was even more shear sensitive. We conclude that dimerization of both PSGL-1 and P-selectin stabilizes tethering and rolling, probably by increasing rebinding within a bond cluster. Because transient tethers may have more than one bond, the mechanical strength of selectin–ligand bonds is likely to be lower than initially estimated. Tether strength may rely more on bond clusters to distribute applied force.

Platelet-derived NO slows thrombus growth on a collagen type III surface

Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an in vitro parallel plate flow system. We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s-1. This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s-1) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s-1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood.

The Synergy Site of Fibronectin Is Required for Strong Interaction with the Platelet Integrin αIIbβ3

Integrins are a class of cell adhesion molecules that bind to ligands containing the RGD peptide sequence. There is increasing evidence that peptide sites other than the RGD site are required for optimal binding of integrins with their ligands. We have examined the sites on the protein fibronectin that are needed for optimal binding to the platelet integrin αIIbβ3 using a strategy of site directed mutagenesis. Single amino acids near the RGD site or near the synergy site of fibronectin were mutated and the resultant proteins were expressed in a bacterial expression system. The purified protein was coated onto glass cover slips. Platelets, expressing αIIbβ3 were perfused over the surface at physiologically relevant shear rates and the extent of adhesion was quantified. We found that the single amino acid substitution of the aspartic acid in the RGD sequence, D1495A, completely abolished adhesion. Surprisingly, the mutants R1445A and R1448Q that are near the RGD site also abolished adhesion of platelets under flow. Additionally, the synergy site mutants R1371A, R1374Q, or R1379A displayed only minimal adhesion of platelets. These results show that the binding site for αIIbβ3 on fibronectin extends over a considerable distance from the RGD site and that these distant sites are required for optimal attachment of cells in the presence of physiologically relevant shear stress.

Teachers in an Interdisciplinary Learning Community: Engaging, Integrating, and Strengthening K-12 Education

This study examines the inputs (processes and strategies) and outputs (perceptions, skill development, classroom transfer, disciplinary integration, social networking, and community development) of a yearlong, interdisciplinary teacher learning and development experience. Eleven secondary math and science teachers partnered with an interdisciplinary team of university engineering mentors in a yearlong engineering education and project implementation program. It consisted of a 6-week on-site resident professional development and collaboration experience, with an ongoing support and follow-up including digital systems. Mixed-method, multisource data indicate that teachers engaged with motivations combining personal, intrinsic interest and classroom integration goals. They formed and sustained an active community of learning and practice that supported their success, on-site and through classroom integration, thereby promoting innovations. Teachers reported positive perceptions throughout the program and demonstrated significant, productive trajectories of change-over-time. Teachers learned and transferred task-specific engineering and scientific skills, as well as more general inquiry-based pedagogical strategies to their secondary classrooms.

Designing and evaluating a STEM teacher learning opportunity in the research university

This study examines the design and evaluation strategies for a year-long teacher learning and development experience, including their effectiveness, efficiency and recommendations for strategic redesign. Design characteristics include programmatic features and outcomes: cognitive, affective and motivational processes; interpersonal and social development; and performance activities. Program participants were secondary math and science teachers, partnered with engineering faculty mentors, in a research university-based education and support program. Data from multiple sources demonstrated strengths and weaknesses in design of the programs learning environment, including: face-to-face and via digital tools; on-site and distance community interactions; and strategic evaluation tools and systems. Implications are considered for the strategic design and evaluation of similar grant-funded research experiences intended to support teacher learning, development and transfer.

Properties of porcine adipose-derived stem cells and their applications in preclinical models

ABSTRACT Adipose-derived stem cells represent a reliable adult stem cell source thanks to their abundance, straightforward isolation, and broad differentiation abilities. Consequently, human adipose-derived stem cells (hASCs) have been used in vitro for several innovative cellular therapy and regenerative medicine applications. However, the translation of a novel technology from the laboratory to the clinic requires first to evaluate its safety, feasibility, and potential efficacy through preclinical studies in animals. The anatomy and physiology of pigs and humans are very similar, establishing pigs as an attractive and popular large animal model for preclinical studies. Knowledge of the properties of porcine adipose-derived stem cells (pASCs) used in preclinical studies is critical for their success. While hASCs have been extensively studied this past decade, only a handful of reports relate to pASCs. The aim of this concise review is to summarize the current findings about the isolation of pASCs, their culture, proliferation, and immunophenotype. The differentiation abilities of pASCs and their applications in porcine preclinical models will also be reported.

Situating teachers’ developmental engineering experiences in an inquiry-based, laboratory learning environment

Many secondary math and science teachers don’t understand the nature and application of engineering adequately to transfer that understanding to their students. Research is needed that investigates and illuminates the process and characteristics of development that addresses this gap. This mixed-method study examines the developmental experiences of 16 public math and science teachers in a yearlong engineering experience. Teachers partnered with university engineering professor-mentors for six weeks on campus followed by ongoing support for transfer to their secondary classes (grades 9–12). Teachers gained engineering knowledge and skills, and an understanding of engineering’s diversity and interdisciplinary nature, plus integrative applied strategies for math and science instruction. They developed productive perceptions (of value, utility, benefits, feasibility and fit) of engineering for their grade 9–12 students, along with efficacy and intent to use what they had learned. Teachers identified positive, profound shifts in their perceptions of engineering and engineers. All teachers integrated engineering principles into their classrooms, but the process was more challenging for math teachers than for science teachers. This developmental experience may help bridge gaps of school-to-life and disciplinary boundaries that separate formal educational environments from authentic life experience.