Barry A. Wolitzky

Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins.

The initial rolling interaction of leukocytes with the blood vessel wall during leukocyte trafficking has been postulated to rely on members of the selectin family of adhesion molecules. Two selectins, E-selectin and P-selectin, have been identified that are expressed on activated endothelial cells. Mice deficient in E-selectin expression have been produced in order to examine the role of this selectin in leukocyte trafficking. Mice homozygous for an E-selectin null mutation were viable and exhibited no obvious developmental alterations. E-selectin-deficient mice displayed no significant change in the trafficking of neutrophils in several models of inflammation. However, blocking both endothelial selectins by treatment of the E-selectin-deficient animals with an anti-murine P-selectin antibody, 5H1, significantly inhibited neutrophil emigration in two distinct models of inflammation. While neutrophil accumulation at early times during thioglycollate-induced peritonitis was dependent on P-selectin, neutrophil accumulation at later time points was blocked by 5H1 only in E-selectin-deficient mice but not in wild-type mice. Similarly, edema as well as leukocyte accumulation in a model of delayed-type hypersensitivity in the skin was almost completely prevented by blockade of P-selectin function with 5H1 in the E-selectin-deficient mice while the same treatment had no effect in wild-type mice. These data demonstrate that the majority of neutrophil migration in both models requires an endothelial selectin but that E-selectin and P-selectin are functionally redundant. These data have important implications in the use of selectin antagonists in the treatment of inflammatory disease.

Heterogeneity of Expression of E- and P-Selectins In Vivo

A novel technique involving radiolabeled monoclonal antibodies was used to characterize and compare the expression of E- and P-selectin on unstimulated, histamine-challenged, and endotoxin-challenged endothelial cells in various tissues of the mouse. Under unstimulated conditions, E-selectin was absent in all organs, but significant expression of P-selectin was observed in several organs. Histamine induced a rapid time-dependent upregulation of P-selectin, with the largest responses observed in mesentery and lung. Significant fold elevations in P-selectin expression occurred as early as 5 minutes after the histamine injection and remained elevated up to 1 hour. Histamine-induced P-selectin upregulation was inhibited by the H1 receptor antagonist diphenhydramine, whereas the H2 receptor antagonist cimetidine had no effect. Endotoxin (lipopolysaccharide [LPS]) also induced a time-dependent expression of P-selectin that reached a maximum between 4 and 8 hours after endotoxin administration. LPS-induced upregulation of P-selectin was greatest in heart and stomach, which exhibited insignificant constitutive expression of P-selectin. LPS also induced a time-dependent upregulation of E-selectin, with maximal expression occurring 3 to 5 hours after intraperitoneal administration. The lung and small intestine exhibited the largest responses to LPS challenge. Histamine administration did not affect E-selectin expression in any tissue. E- and P-selectin-deficient mice were used to test the specificity of monoclonal antibody binding in unstimulated, histamine-challenged, and LPS-stimulated tissues. Vascular binding of the radiolabeled E-selectin and P-selectin monoclonal antibodies was not observed in the respective deficient mice. These findings suggest that P-selectin is constitutively expressed on vascular endothelium in some tissues of the mouse and that there are significant regional differences in the magnitude and time course of histamine- and endotoxin-induced P-selectin expression. In contrast, E-selectin appears to be absent on unstimulated vascular endothelium but is upregulated within 3 hours after the administration of endotoxin in most tissues.

Liver endothelial E‐selectin mediates carcinoma cell adhesion and promotes liver metastasis

E‐selectin is a cytokine‐inducible endothelial cell adhesion receptor which is involved in the process of leukocyte rolling, the first in a cascade of interactions leading to leukocyte transmigration. Several studies have implicated this receptor in carcinoma cell adhesion to the endothelium, an interaction thought to be required for tumor extravasation during metastasis. To study the role of this receptor in the process of metastasis, we utilized a murine carcinoma line H‐59 which is highly metastatic to the liver in vivo. When adhesion of H‐59 cells to primary cultures of murine hepatic endothelial cells was measured, it was found that the tumor cells had a low which basall of adhesion to the sinusoidal endothelial cells, which could be significantly and specifically augmented by pre‐activation of the endothelial cells with rTNFα. This incremental increase in adhesion to the activated endothelium could be completely and specifically abolished by a neutralizing monoclonal antibody to murine E‐selectin (MAb 9A9). Similar results were obtained with 2 highly metastatic human colorectal carcinoma lines, HM 7 and CX‐1, but not with a second murine subline, M‐27, which is poorly metastatic to the liver. To assess the role of E‐selectin in metastasis to the liver in vivo, the effect of MAb 9A9 on experimental liver metastasis was evaluated using the syngeneic H‐59 model. We show here that this antibody caused a marked, specific and Fc‐independent inhibition of experimental liver metastasis, reducing the median number of metastases by 97% relative to the control groups. Our results provide evidence that endothelial E‐selectin is a mediator of carcinoma metastasis to the liver. Int. J. Cancer 71:612‐619, 1997.

Endothelial cell adhesion molecule expression in gene-targeted mice

Gene-targeted mice are now routinely employed as tools for defining the contribution of different leukocyte and endothelial cell adhesion molecules to the leukocyte recruitment and tissue injury associated with acute and chronic inflammation. The objective of this study was to determine whether gene-targeted mice that are deficient in CD11/CD18, intracellular adhesion molecule-1 (ICAM-1), or P-selectin exhibit an altered constitutive or induced expression of the endothelial cell adhesion molecules E- and P-selectin. The gene-targeted mice were all developed in the 129Sv mouse strain and backcrossed into C57Bl/6J mice. The number of backcrosses ranged between 8 (P-selectin) and 10 (CD18 and ICAM-1) generations. The dual-radiolabeled monoclonal antibody technique was used to quantify E- and P-selectin expression in different vascular beds. In the unstimulated state, E-selectin expression was significantly elevated (relative to wild-type mice) in the stomach, large intestine, and brain of mutants deficient in ICAM-1. In general, constitutive expression of P-selectin did not differ between wild-type, ICAM-1-deficient, and CD11/CD18-deficient mutants. In CD11/CD18-deficient mice, tumor necrosis factor-α (TNF-α) administration elicited a more profound upregulation of P-selectin in several vascular beds, compared with wild-type and ICAM-1-deficient mice. E-selectin expression in brain of TNF-α-stimulated, ICAM-1-deficient, and P-selectin-deficient mice was attenuated compared with wild-type mice. These findings indicate that chronic deficiency of some of the adhesion glycoproteins that mediate leukocyte recruitment alters basal and induced surface expression of other adhesion molecules on endothelial cells.

N-Benzylpyroglutamyl-l-phenylalanine derivatives as VCAM/VLA-4 antagonists

A series of N-(N-benzylpyroglutamyl)-4-substituted-L-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. Analogues substituted by electron deficient benzoylamino groups bearing bulky ortho substituents had low-nM potency in an ELISA assay and low-microM activity in a cell based assay.

Chapter 8 Differential Subunit and Isoform Expression Involved in Regulation of Sodium Pump in Skeletal Muscle

Publisher Summary In a variety of cellular systems, there is evidence that a rise in intracellular sodium ion concentration leads to up-regulation of the sodium pump. Because of their many experimental advantages, tissue culture systems have played a major role in the development and testing of this concept. The rationale was that, as a consequence of sodium pump inhibition, intracellular sodium would rise, triggering up-regulation. Recent work by Pressley and colleagues (1986), using ARL 15 cells, has provided perhaps the most rigorous documentation of the ion changes and time course of up-regulation. Other conditions that should lead to a rise in intracellular sodium-for example, reduction in extracellular potassium ion concentration, likewise result in up-regulation of the sodium pump. For excitable cells, the influx of sodium ions can be augmented by opening the voltage-sensitive sodium channels in their plasma membranes with pharmacological agents. As an offshoot of this work, to the chapter focuses on expression systems in which selected sodium pump genes can be expressed and studies the behavior of the resultant subunits.

The design and synthesis of potent cyclic peptide VCAM–VLA-4 antagonists incorporating an achiral Asp-Pro mimetic

The Asp-Pro sequence of the cyclic peptide Ac-HN-Tyr-Cys*-Asp-Pro-Cys*-OH (1) could be replaced with the achiral dipeptide mimetic 1-(2-aminoethyl)cyclpentylcarboxylic acid with retention of potent inhibition of the VCAM-VLA-4 interaction.

Imide and lactam derivatives of N-benzylpyroglutamyl-l-phenylalanine as VCAM/VLA-4 antagonists

A series of imides and lactams derived from 4-amino-N-benzylpyroglutamyl-L-phenylalanine was prepared and evaluated for activity as VCAM/VLA-4 antagonists. Imides were more potent than the corresponding lactams; several had subnanomolar IC50s in an ELISA based assay and were also highly effective at blocking VLA-4 expressing Ramos cell binding to VCAM coated plates.

Cyclic thioether peptide mimetics as VCAM–VLA-4 antagonists

Selective substitution of a sulfur atom by carbon in a highly potent 13-membered cyclic disulfide was accomplished by intramolecular displacement of a bromide. The potency of the resulting thioethers in the VCAM/VLA-4 assay was dependent on ring size and the position of the sulfur atom.

N-Acyl Phenylalanine Analogues as Potent Small Molecule VLA-4 Antagonists

We have identified a series of low molecular weight (Mr < 500) N-acylphenylalanines that are effective inhibitors of the VCAM-VLA-4 interaction. Investigation of the SAR of the N-acyl moiety led to the identification of N-benzylpyroglutamyl derivatives as being particularly potent.