Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.

Elevated levels of circulating chromogranin A (CgA), a protein stored in the secretory granules of many neuroendocrine cells and neurons, have been detected in the blood of patients with neuroendocrine tumors or heart failure. The pathophysiological role of increased secretion of CgA is unknown. Using mice bearing subcutaneous tumors genetically engineered to secrete CgA in circulation, we have found that increased blood levels of this protein prevent vascular leakage induced by tumor necrosis factor‐α (TNF) in the liver venous system. Structure–activity studies, carried out with CgA fragments administered to normal mice, showed that an active site is located within residues 7–57 of CgA. Accordingly, an anti‐CgA antibody directed to residues 53–57 inhibited the effect of circulating CgA, either endogenously produced or exogenously administered, on liver vessels. Studies of the mechanism of action showed that CgA inhibits TNF‐induced VE‐cadherin down‐regulation and barrier alteration of cultured endothelial cells, in an indirect manner. Other effectors, such as thrombin and vascular endothelial growth factor were partially inhibited by CgA N‐terminal fragments in in vitro permeability assays. These findings suggest that circulating CgA could help regulate the endothelial barrier function and to protect vessels against TNF‐induced plasma leakage in pathological conditions characterized by increased production of TNF and CgA, such as cancer or heart failure.

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.

Angiogenesis, the formation of blood vessels from pre-existing vasculature, is regulated by a complex interplay of anti and proangiogenic factors. We found that physiologic levels of circulating chromogranin A (CgA), a protein secreted by the neuroendocrine system, can inhibit angiogenesis in various in vitro and in vivo experimental models. Structure-activity studies showed that a functional anti-angiogenic site is located in the C-terminal region, whereas a latent anti-angiogenic site, activated by cleavage of Q76-K77 bond, is present in the N-terminal domain. Cleavage of CgA by thrombin abrogated its anti-angiogenic activity and generated fragments (lacking the C-terminal region) endowed of potent proangiogenic activity. Hematologic studies showed that biologically relevant levels of forms of full-length CgA and CgA1-76 (anti-angiogenic) and lower levels of fragments lacking the C-terminal region (proangiogenic) are present in circulation in healthy subjects. Blood coagulation caused, in a thrombin-dependent manner, almost complete conversion of CgA into fragments lacking the C-terminal region. These results suggest that the CgA-related circulating polypeptides form a balance of anti and proangiogenic factors tightlyregulated byproteolysis. Thrombin-induced alteration of this balance could provide a novel mechanism for triggering angiogenesis in pathophysiologic conditions characterized by prothrombin activation.

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.

We have investigated the role of p55 and p75 tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2, respectively) in TNF-induced alteration of endothelial permeability in vitro and in vivo. Stimulation of TNFR1 with an agonist antibody or a receptor-selective TNF mutein increased the flux of¹²⁵I-albumin through endothelial cell monolayers. An antagonist anti-TNFR1 antibody, but not antagonist anti-TNFR2 antibodies, blocked the activity of TNF in vitro. Stimulation of TNFR1, but not TNFR2, induced cytoskeletal reorganization associated with increased permeability. SB-203580, a p38 mitogen-activated protein kinase inhibitor, blocked TNFR1-induced cytoskeletal reorganization and permeability. A selective mouse TNFR1 agonist and human TNF, which binds to murine TNFR1, increased the leakage of trypan blue-albumin from liver vessels in mice. These results indicate that stimulation of TNFR1 is necessary and sufficient to increase endothelial permeability in vitro and in vivo. However, an antagonist anti-murine TNFR2 antibody partially inhibited the effect of murine TNF on liver vessels, suggesting that TNFR2 also plays a role in the regulation of TNF-induced vascular permeability in vivo.