Here we have studied the involvement of endothelial heparan sulfate in inflammation by inactivating the enzyme N-acetyl glucosamine N-deacetylase-N-sulfotransferase-1 in endothelial cells and leukocytes, which is required for the addition of sulfate to the heparin sulfate chains. Mutant mice developed normally but showed impaired neutrophil infiltration in various inflammation models. These effects were due to changes in heparan sulfate specifically in endothelial cells. Decreased neutrophil infiltration was partially due to altered rolling velocity correlated with weaker binding of L-selectin to endothelial cells. Chemokine transcytosis across endothelial cells and presentation on the cell surface were also reduced, resulting in decreased neutrophil firm adhesion and migration. Thus, endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endothelium.

Understanding cellular and molecular mechanisms of tumor metastasis is critically important for the development of new approaches to cancer treatment. One of the rate-limiting steps in metastatic dissemination is the adhesion of circulating cancer cells to the microvascular endothelium (for review, see Ref. 1). Recent experimental evidence identified endotheliumattached blood-born tumor cells as the seeds of secondary tumors (2). In the lung, early metastatic colonies were entirely within the blood vessels, and hematogenous metastases originated from the intravascular proliferation of tumor cells anchored to the endothelia (2). These results underscored the significance of intravascular intercellular adhesion in cancer metastasis.Although there is a substantial body of evidence demonstrating the role of various adhesion molecules in tumor cell adhesion (1, 3), the molecular and cellular mechanisms of cancer cell adhesion are still often modeled after the dynamics of the leukocyte adhesion cascade. Despite the many physical similarities, interaction of leukocytes and circulating malignant cells with the vascular endothelium are likely to be driven by distinct molecular mechanisms. For example, it is well documented that under conditions of shear force, circulating leukocytes participate in a multi-step cascade of sequential adhesion events involving rolling, adhesion, and transmigration across the vascular wall, where rolling is the first and rate-limiting step ultimately required for stable leukocyte adhesion to the endothelial cells (EC) 1 (4). However, in contrast to leukocytes, published data regarding the rolling and adhesion of tumor cells on vascular endothelium suggest a non-leukocyte-like mechanism (5-9). Furthermore, it is also not clear whether this step is required for stable adhesion of tumor cells to the endothelium. Leukocyte rolling is mostly mediated by the interaction of the members of C-type lectin family, selectins, with their cognate carbohydrate ligands (4, 10). Studies from our laboratories (11, 12) as well as other investigators (13) have recently shown that another lectin, galectin-3, plays a key role in initiating the adhesion of human breast and prostate cancer cells to the endothelium by specifically interacting with the cancerassociated carbohydrate, T antigen. However, these studies were carried out under static conditions, and the relevance of galectin-3-T antigen interactions in mediating cancer cell adhesion under conditions of flow has not been investigated.Shear forces have an important influence on cell adhesion and other cellular functions, and malignant cell lines appear to possess different adhesive properties under static and dynamic conditions (14,15). To elucidate the molecular mechanisms of intercellular adhesive interactions relevant to breast cancer metastasis, we examined the adhesive behavior of two human breast carcinoma cell lines exhibiting distinct metastatic po-

In leukocytes, C3a and C5a cause chemotaxis in a Gi-dependent, pertussis toxin (PT)-sensitive fashion. Because we found that HUVECs and immortalized human dermal microvascular endothelial cells express small numbers of C3aRs and C5aRs, we asked what the function of these receptors was on these cells. Activation of the C3aR caused transient formation of actin stress fibers, which was not PT-sensitive, but depended on rho activation implying coupling to Gα12 or Gα13. Activation of the C5aR caused a delayed and sustained cytoskeletal response, which was blocked by PT, and resulted in cell retraction, increased paracellular permeability, and facilitated eosinophil transmigration. C5a, but not C3a, was chemotactic for human immortalized dermal microvascular endothelial cells. The response to C5a was blocked by inhibitors of phosphatidylinositol-3-kinase, src kinase, and of the epidermal growth factor (EGF) receptor (EGFR) as well as by neutralizing Abs against the EGFR and heparin-binding EGF-like factor. Furthermore, immune precipitations showed that the EGFR was phosphorylated following stimulation with C5a. The C5aR in endothelial cells thus uses a signaling cascade–transactivation of the EGFR–that does not exist in leukocytes, while the C3aR couples to a different G protein, presumably Gα12/13.