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Chromogranin A protects vessels against tumor necrosis factor -induced vascular leakage ¹

Department Biological and Technological Research (DIBIT)-San Raffaele H. Scientific Institute, Milan; and
^* Institute of General Pathology and Center for Studies on Cellular Pathology of the CNR, Milan, Italy

²Correspondence: Department Biological and Technological Research (DIBIT), San Raffaele H Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. E-mail: elisabetta.ferrero@hsr.it or angelo.corti{at}hsr.it

SPECIFIC AIMS

Elevated levels of soluble tumor necrosis factor (TNF) receptors and 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 aim of this study was to investigate whether endogenously produced CgA could affect TNF-induced vascular leakage.

PRINCIPAL FINDINGS

1. Increased blood levels of CgA prevented TNF-induced vascular leakage in the liver of mice bearing subcutaneous tumors genetically engineered to secrete CgA in circulation (Fig. 1 ).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of endogenous CgA and of anti-CgA neutralizing mAb 5A8 on TNF-induced leakage of trypan blue albumin from liver vessels in tumor-bearing (a) and normal mice (b). Normal BALB/c mice and CD-1 nu/nu BR mice bearing CgA-secreting (RMA-3A6) and CgA nonsecreting (RMA-WT) tumors were injected intraperitoneally with the reagents indicated below each panel 30 min before liver perfusion with trypan blue albumin. The following doses were used: TNF (2 ng), anti-CgA mAb 5A8 (30 µg), or control mAb 9E10 (30 ng) in sterile 0.9% sodium chloride. Controls ("None") received 0.9% sodium chloride. Each bar represents the mean ± SD of 3 mice.

2. 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.

3. 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.

4. 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.

5. Other effectors, such as thrombin and vascular endothelial growth factor, were partially inhibited by CgA amino-terminal fragments in in vitro permeability assays.

CONCLUSIONS AND SIGNIFICANCE

Significance of the main findings
CgA is a secretory protein stored in the dense core granules of the adrenal medulla and of many other neuroendocrine cells and neurons. After an appropriate stimulus, CgA is released into the extracellular environment together with coresident hormones. Elevated levels of circulating CgA have been detected in the blood of patients with endocrine and neuroendocrine tumors, hepatic failure, and renal failure. We recently showed that circulating CgA is markedly increased in patients with heart failure, depending on the severity of the diseases, and that it correlates with TNF and TNF-soluble receptors.

Results of this study show that increased levels of circulating CgA can inhibit TNF-induced leakage of solutes from liver vessels in mouse models. These findings suggest that CgA contributes to the regulation of vessel barrier function in those pathological conditions characterized by increased secretion of TNF and CgA, such as heart failure or (neuro)endocrine tumors. Given that CgA is concentrated and stored within secretory granules and rapidly released after an appropriate neural or hormonal stimulus, this protein could be an important part of neuroendocrine mechanisms that control the proinflammatory activity of TNF.

The concentration of CgA in neuroendocrine secretory vesicles is very high, approaching millimolar levels. Thus, after secretion, CgA could affect endothelial cells by paracrine mechanisms, considering the relatively high concentration in the secretory cell microenvironment. CgA is also present in the blood of normal subjects at 0.02–0.09 µg/mL and up to 1–2 µg/mL in patients with heart failure or neuroendocrine tumors. Since in our in vitro and in vivo assays we observed TNF inhibition at concentrations ranging from 0.1 to 3 µg/mL, it is possible that pathological concentrations of circulating CgA are functionally active at a systemic level. This hypothesis is supported by results with mice bearing subcutaneous mouse lymphomas we had genetically engineered to express and secrete human CgA in the blood stream. In these models we observed that TNF-induced leakage of macromolecules in the liver is inhibited in mice bearing CgA-secreting tumors but not in nonsecreting tumor-bearing mice (Fig. 1) . A neutralizing anti-CgA antibody increased the TNF-induced leakage in mice bearing CgA-secreting tumors, but not in normal mice. These results point to systemic effects of CgA only in conditions of increased production, as it may occur in patients with neuroendocrine tumors or heart failure (see Fig. 2 ).

View larger version (32K): [in this window] [in a new window]   Figure 2. Schematic diagram of the main findings of the study and their interpretation. In conditions of hyperproduction of CgA as may occur pathologically (e.g., heart failure or neuroendocrine tumors), circulating CgA could inhibit TNF-induced vascular leakage.

Circulating CgA and soluble TNF receptors are important prognostic markers in patients with heart failure. Although the clinical significance of TNF production in heart failure patients remains uncertain, its ability to induce cachexia, left ventricular dysfunction, and pulmonary edema suggests that TNF could play a pathogenetic role. It is possible that the regulated secretion of CgA, in concert with TNF-soluble receptors, contribute to reduce the potentially dangerous effects of pathological levels of TNF.

Structure–activity studies
The results of structure–function studies carried out with natural, recombinant and synthetic fragments indicate that the amino-terminal region of CgA_7-57 contains the active site. In earlier work we showed that CgA_7-57 can induce fibroblast adhesion to solid phases. Region 47-57 was found to contain a site critical for fibroblast adhesion. The importance of the region 47-57 for the biological activity of CgA is reflected by the high degree of homology between human, rat, mouse, porcine, ostrich, and frog CgA, compared with other regions. mAb 5A8, a monoclonal antibody directed to residues Arg⁵³, His⁵⁴, and Leu⁵⁷, blocks the activity of CgA on vascular leakage as well as the proadhesive activity of CgA amino-terminal fragments on fibroblasts. Other studies showed that CgA amino-terminal fragments inhibit the myogenic tone in isolated conduit vessels and resistance arteries and exert negative inotropic effects. However, whether these effects are mediated by the same site remains to be investigated.

Mechanism of action
We investigated the mechanism of action and the cellular targets of CgA/TNF in vascular leakage. It is known that TNF can induce vascular leakage by interacting with the p55 TNF receptor on endothelial cells. Thus, it is possible that the endothelial lining of vessels is the primary site of action also of CgA. Using cultured HUVEC monolayers, we observed that CgA and its amino-terminal fragments can inhibit TNF-induced cytoskeletal reorganization, intercellular VE-cadherin down-modulation (a transmembrane protein important for cell–cell adhesion) and barrier alteration, suggesting that the endothelial lining of vessels can indeed be affected by CgA. Our study suggest that CgA does not act by blocking the TNF/TNF receptor interactions. More likely, CgA interacts with other components of endothelial cells. It has been reported that bovine aorta endothelial cells bind and internalize 1 nM ¹²⁵I-labeled CgA; both the binding and internalization are temperature and time dependent, reaching a maximum after 2 h of incubation. Although a specific receptor was not identified, this study showed that endothelial cells can bind CgA with low affinity and high capacity. The results of confocal microscopy studies showed that HUVEC internalize CgA_1-78. Although internalization is not necessarily linked to changes in permeability, this result supports the concept that CgA can indeed interact with endothelial cells.

We have also found that CgA_1-78 could partially inhibit thrombin-induced and VEGF-induced permeability of HUVEC. This may have several explanations: CgA could act on signaling components of endothelial cells common to TNF and these effectors. Alternatively, these phenomena are the result of enhanced surface expression or function of adhesion molecules (e.g., VE-cadherin) in a manner that cannot be overcome by TNF, thrombin, or VEGF signaling. The physiological relevance of this finding remains to be established, as we failed to demonstrate inhibition of VEGF permeability in our in vivo model. Other factors likely are critical for the differential regulation of TNF, VEGF, thrombin, and CgA activity in vivo.

CONCLUSIONS

In conclusion, we have found that increased levels of circulating CgA protect vessels from TNF-induced vascular leakage in animal models. We propose that increased (neuro)endocrine secretion of CgA could contribute to protect vessels against TNF-induced plasma leakage in pathological conditions characterized by increased production of TNF and CgA, such as in patients with heart failure and cancer (see Fig. 2 ). This work could stimulate further studies aimed at assessing whether exogenously administered CgA can reduce the vascular leakage in inflammatory diseases associated with overproduction of TNF.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0922fje;

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