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.

A growing body of evidence suggests that chromogranin A (CgA), a secretory protein released by many neuroendocrine cells and frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors, is a precursor of several bioactive fragments. This work was undertaken to assess whether the N‐terminal fragment CgA _1–76 (called vasostatin I) can inhibit the proangiogenic activity of vascular endothelial growth factor (VEGF), a factor involved in tumor growth. The effect of recom‐binant human vasostatin I (VS‐1) on VEGF‐induced human umbilical endothelial cells (HUVEC) signaling, proliferation, migration, and organization has been investigated. We have found that VS‐1 (3 μg/ml;330 nM) can inhibit VEGF‐induced ERK phosphorylation, as well as cell migration, proliferation, morphogenesis, and invasion of collagen gels in various in vitro assays. In addition, VS‐1 could inhibit the formation of capillary‐like structures in Matrigel plugs in a rat model. VS‐1 could also inhibit basal ERK phosphorylation and motility of HUVEC, leading to a more quiescent state in the absence of VEGF, without inducing apoptotic or necrotic effects. Conclusion: These findings suggest that vasostatin I may play a novel role as a regulator of endothelial cell function and homeostasis.—Belloni, D., Scabini, S., Foglieni, C., Veschini, L., Giazzon, A., Colombo, B., Fulgenzi, A., Helle, K. B., Ferrero, M. E., Corti, A., Ferrero, E. The vasostatin‐I fragment of chromogranin A inhibits VEGF‐induced endothelial cell proliferation and migration. FASEB J. 21, 3052–3062 (2007)

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.

Angiogenesis is a complex, orchestrated process that plays a critical role in several conditions and has special relevance in the progression of cancer. Hypoxia is the major stimulus for angiogenesis, and hypoxia-inducible transcription factor–1 alpha (HIF-1α) is its key mediator. We set up a novel in vitro model of HIF-1α up-regulation by treating human umbilical vein endothelial cells (HUVECs) with the hypoxia-mimicking deferoxamine (DFO) and found that this condition was sufficient to promote angiogenesis, like the well-known HUVEC model cultured under low pO_2. The proteasome inhibitor bortezomib, which induces strong apoptosis in cancer cells, abrogated proliferation and angiogenesis of HUVECs when used at a high concentration (100 nM), yet promoted both functions at a low dosage (10 nM). This double-edged effect appeared to be mediated by differential effects exerted by the different concentrations of bortezomib on 2 master regulators of tumor-associated angiogenesis, HIF-1α and nuclear factor kappa B (NF-kB). Significantly, when HUVECs were induced to express HIF-1α prior to bortezomib treatment, proliferative and angiogenic responses were abolished, and a greatly enhanced proapoptotic effect was promoted with both concentrations of the drug. These findings indicate that HIF-1α up-regulation may sensitize endothelial cells to the antiangiogenic and proapoptotic effects of bortezomib and might be exploited to target tumor-associated vessels in the course of antiangiogenic therapies.

Amino-biphosphonates (like pamidronate) activate human Vγ9/Vδ2 T lymphocytes and promote their cytotoxicity against multiple myeloma cells. T-cell receptor (TCR)–mediated effector functions of γδ cells are enhanced upon triggering of the activating receptor NKG2D by MICA, a stress-inducible antigen expressed by epithelial and some hematopoietic tumors, including multiple myeloma. Here we show that MICA was expressed not only by myeloma cell lines and by 6 of 10 primary multiple myeloma cells from patients but also by bone marrow plasma cells from all (six of six) patients with preneoplastic gammopathy (monoclonal gammopathy of undetermined significance, MGUS), a direct precursor of multiple myeloma. Moreover, compared with multiple myeloma plasma cells, MICA was expressed by MGUS plasma cells at significantly (P < 0.05) higher levels. MICA expressed by myeloma cell lines contributed to killing and IFN-γ production by Vγ9/Vδ2 cells only upon pamidronate treatment, suggesting a dual interaction between Vγ9/Vδ2 lymphocytes and multiple myeloma plasma cells involving both TCR triggering and NKG2D-mediated signals. Finally, MICA enhanced killing of freshly derived, pamidronate-treated multiple myeloma cells from patients by γδ cells, as indicated by the significantly (P < 0.05) higher γδ cytotoxicity against MICA-positive rather than MICA-negative multiple myeloma cells. Our results indicate that MICA expressed by monoclonal plasma cells is functional and correlates with disease stages, suggesting a role for the molecule in the immune surveillance against multiple myeloma. Moreover, pamidronate-activated Vγ9/Vδ2 lymphocytes can be exploited in the immune therapy of early stages multiple myeloma and possibly of premalignant disease.