Patent Application
20110281374
The present invention relates to use of Interleukin-1 alpha (IL-1α) as a serum or plasma biomarker for arterial aneurysm, especially abdominal aortic aneurysm (AAA).
Arterial aneurysm (referred to herein as AAA in relation to the disease associated with the aorta, i.e. abdominal aortic aneurysm, but relevant to other arterial vessels) is pathological ballooning of the artery, which is defined as a focal dilation of the artery generally exceeding 150% of normal diameter (Johnston et al. ‘Suggested standards for reporting on arterial aneurysms’, J. Vascular Surg. (1991) 13, 452-458). AAA is thought to occur in about 2-13% of the adult population and rupture of AAA is a significant clinical problem in the elderly with about 1% of men over 65 thought to suffer from ruptured AAA with an associated mortality of greater than 70%. There is currently no comprehensive screening strategy for AAA; individuals with significant disease are usually identified during investigation for other conditions.
The cellular and molecular pathology of AAA is poorly understood. However, it is probable that aortic dilation progresses from the inappropriate remodelling of the vessel wall in response to the chronic inflammatory process within the artery. Certainly, there is significant inflammatory infiltrate into the vessel wall and the secretion of matrix metalloproteinases (MMPs) such as MMP-9 by monocyte-macrophages may contribute to loss and for disorganisation of the structural elastic lamina which support the arterial architecture (Gong et al., J. Clin Invest. (2008) 118, 3012-3024; Carmeliet et al. J. Clin. Invest. (2000) 105, 1519-1520). The cellular complexity of the aneurysm is substantially increased by the deposition of a mural thrombus which is rich in neutrophilic granulocytes (Houard et al. ‘Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophil-derived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus’ FASEB J. (2009) 23, 1376-1383). Nevertheless, much remains unknown about the molecular mechanisms which initiate or support the progression of AAA, although risk factors include the male gender, smoking and family history.
IL-1α has been previously implicated as having a role as an inflammatory factor in the development of both atherosclerosis and AAA, but neither in atherosclerosis nor AAA has this been linked with measureable presence as a serum factor. Rather in relation to atherosclerosis, there has been much interest in correlation between reduced risk of atherosclerosis and atherosclerosis-related disorders and high titres of IL-1α auto-antibodies (Published International Applications WO 2007/015128 and WO 2007/132338 of Xbiotech, Inc.). The reason for such auto-antibodies has not been elucidated, but they have also been reported in sera of apparently healthy humans, older men being found to have the highest titres. Attempts to measure IL-1α in such samples led to the conclusion that IL-1α molecules are usually inaccessible for immunometric assay (Hanson et al. Eur. J. Clin. Invest. (1994) 24, 212-218).
The majority of pro-IL-1α is found in the plasma membrane or the nucleus of cells (W. P. Arend, Cytokine and Growth Factor Reviews (2008) 13, 323-340). Mature IL-1α is known to be released by the enzyme calpain and binds to the IL-1 receptor resulting in translocation of the transcription factor NF-kB to the nucleus. More recently, IL-1α has been found to be expressed on the minor sub-set of monocytes which also coexpress CD14 and CD16 (Published International Application no. 2010/030979 of Xbiotech, Inc), but such monocytes have not been linked to measureable serum IL-1α in AAA patients In Lindeman et al. Clin Sci. (2008) 114, 687-697, it is reported that IL-1α mRNA is increased in aneurysmal wall samples compared to atherosclerotic wall samples, but no results are given for IL-1α protein. Hence, while IL-1α has been implicated in the inflammatory process associated with development of AAA, it has not previously been recognised as having any value as a serum biomarker for that condition.
That this is indeed the case was an unexpected finding of the inventors from carrying out studies aimed at resolving whether endovascular aneurysm repair using stent grafts (EVAR), now generally favoured over open surgical repair (OSR) for treatment of AAA, reduces the systemic inflammatory response, even though it leaves a substantial volume of diseased tissue and mural thrombus in situ.
The inventors have established that IL-1α is elevated in serum of pre-operative AAA patients but that EVAR causes significant reduction in serum level of IL-1α by 6 months. This mirrors a similar pattern seen with IL-8, a cytokine which has previously been linked with AAA.
Hence, the present invention provides a method of diagnosing or determining the degree of an arterial aneurysm, especially an abdominal aortic aneurysm (AAA), which comprises determining the presence or level of IL-1α in a serum or plasma sample.
That such measurement of IL-1α has physiological relevance in relation to the development of AAA is further supported by the studies of the inventors reported herein which show that serum of pre-operative AAA patients will prime cultured endothelial cells for increased neutrophil recruitment in response to low dose tumour necrosis factor-α (TNF-α) in a flow-based neutrophil adhesion assay, but this response is lost in post-EVAR serum by 6 months; this correlates with reduced serum titre of IL-1α and the addition of functional neutralising antibody against IL-1α, but not IL-8, to pre-operative serum also inhibits neutrophil recruitment in the same assay.
Measurement of Il-1α may be made in more than one sample taken at different time points. Thus, measurement of IL-1α may be made in samples taken at different time points pre- and/or post-operatively to predict, for example, rate of disease progression, likelihood of, or projected time to surgical intervention and/or progress to normalisation post-EVAR. Observation of re-establishment of high titres after EVAR may also be diagnostic of late technical graft failure.
Such reliance on IL-1α as a biomarker will preferably employ an immunoassay for detecting IL-1α. Suitable assays for this purpose are well known. They include double-sandwich ELISA employing for example rabbit polyclonal antibodies specific for recombinant IL-1α as described in Hansen et al. (ibid). A commercially available assay system may be employed, e.g. a Milliplex MAP immunoassay from Milllipore (Millipore, Billerica, Mass., USA) as used for the studies reported herein. This is based on the Luminex bead system and may be conveniently used to assay a variety of analytes of interest simultaneously in a single sample.
Thus it may be desired to measure IL-1α together with one or more further analytes whose presence in serum is known to correlate with risk or progression of AAA, either in the same sample or one or more equivalent samples. These include, for example, IL-8 (Lindeman at al. ibid; Norgren et al. J. Endovascular Surgery 4, 169-173; Parodi et al. J. Endovascular Therapy (2001) 8, 114-124) and secreted metaloproteinases such as MMP-9 as noted above. The studies reported herein further support additional use of IL-8 as biomarker for AAA since reduction of serum IL-8 between pre- and post-EVAR samples was found, although antibody blockade of IL-8 in pre-operative serum had no effect on neutrophil recruitment to TNF-α primed endothelial cells. Monitoring of both IL-1α and IL-8 in serum or plasma, preferably by measurement in the same sample, may be preferred in relation to predicting AAA progression, either alone or as part of data collection for a multi-variate predictive algorithm.
Finding of IL-1α, or both of IL-1α and IL-8, at a serum concentration of at least about 50 pg/ml, e.g. about 50-100 pg/ml, may be taken as indicative of AAA, especially where there has been previous diagnosis of atherosclerosis.
In some circumstances a functional assay for IL-α and for IL-8 may be carried out as well as or instead of an immunoassay.
Detection of IL-1α in accordance with the invention may be supplemented by assessment of aneurysm size by ultrasound or CT scan and/or assessment of burden of mural thrombus by CT scan to aid determination of disease progression and/or necessity for surgical intervention.
The studies reported below provide background to the invention and illustrate the invention by way of exemplification with reference to the following figures.
=rolling;
=firmly adherent;
=transmigrated. There is a significant decrease in the proportion of neutrophils rolling on endothelial cells stimulated with 100 U/ml compared to 5 U/ml TNF, and a significantly higher level of transmigrated neutrophils. *=p<0.05, **p<0.01 by paired t-test respectively; Data are mean±SEM; n=3.
The serum of patients with AAA was screened for the presence of a number of cytokines before and 6 months after EVAR. Patient serum was also utilised to stimulate cultured endothelial cells, which were subsequently tested in a flow-based neutrophil adhesion assay. In such flow assays, pre-operative serum did not directly activate endothelial cells to support neutrophil adhesion unless such cells were exposed to TNF-α. With such priming, there was significant increase in the number of neutrophils recruited into the sub-endothelial environment. In serum collected 6 months after EVAR, both IL-8 and IL-1α were found to be significantly reduced compared to levels seen in pre-operative serum and were normalised to the levels seen in control samples. Moreover, reductions in the concentrations of these cytokines correlated with a loss in the ability of patient serum to cause neutrophil recruitment to TNF-a exposed endothelial cells. As also already noted above, antibody neutralisation of IL-1α in pre-operative serum, but not IL-8, also completely removed the capacity for neutrophil recruitment in the same flow assay.
Seventeen patients with a mean age 80.3 (range 69-88) and who were undergoing elective EVAR, had a mean aneurysm size of 6.9 cm (range 5.4-10). Fourteen patients had Zenith and three had Excluder devices implanted. All patients with AAA were asymptomatic, but one had a contained rupture. Four patients had fenestrated EVAR for juxta-renal abdominal aortic aneurysm. The control cohort consisted of 8 patients with a mean age of 72.5 (range 65-89), with no aortic aneurysm, as proven by computerized tomography (CT) scan performed for other diseases.
Blood samples were collected into vacuette Z Serum Sep Clot Activator tubes (Greiner Bio One) from patients undergoing elective EVAR protocols pre-operatively and 6 months post-operatively. Serum was isolated via centrifugation, aliquoted and stored until use at −80° C.
Milliplex MAP immunoassay was purchased from Millipore (Millipore, Billerica, Mass., USA). This assay is based on the Luminex bead system which can assay over 20 analytes in a small volume (50 μl) using flow cytometery technology. The serum concentration of IL-1-α, IL-1β, IL-4, IL-6, IL-8, IL-10, IFN-γ, IP-10, MCP-1, TNF-α and TNF-β were measured using the luminex assay, carried out according to manufacturers instructions and as previous published (Tull et al. PLOS Biology (2009) e1000177). Serum concentrations were measured on a LX100 machine (Luminex Corp, USA) and calibrated against titrations of recombinant standard for each analyte using STarStation software (ACS, USA).
Human umbilical vein endothelial cells were isolated as previously described (Cooke et al. Microvascular Res. (1993) 45, 33-45) and cultured in M199 (Gibco Invitrogen Compounds, Paisley, Scotland) supplemented with 10 ng/ml epidermal growth factor, 35 μg/ml gentamycin, 1 μg/ml hydrocortisone (all from Sigma, UK), 2.5 μg/ml amphotericin B (Gibco Invitrogen Compounds) and 20% FCS (Sigma). Primary cells were sub-cultured into six channel p-Slide VI flow chambers (Ibidi, Munich, Germany) until confluent. Confluent endothelial cells were cultured for 24 h with medium in which FCS was substituted for 30% serum from patients or aged matched controls. An additional control was endothelial cells cultured continuously in 20% FCS. Endothelial cells were then stimulated with 5 U/ml TNF-a (Sigma, UK) for the final 4 hours of culture before flow assay. In some experiments function neutralising antibodies against IL-1α or IL-8 (10 μg/ml, both from R&D Systems, UK) were added to patient serum prior to addition to culture medium.
Human neutrophils were isolated from the blood of healthy donors by density-gradient centrifugation (Histopaque-1077 and Histopaque-1119; Sigma) and suspended in phosphate buffered saline containing 0.1% bovine serum albumin (Sigma) (PBS/Alb). Six channel μ-Slide VI flow chambers were mounted on a phase contrast video microscope (Inverted Labovert, Leitz).
Differences between individual treatments were evaluated by paired t-test. p<0.05 were considered statistically significant. Variation between multiple treatments was evaluated using ANOVA, followed by Bonferroni's multiple comparison test. Correlation was calculated using GraphPad in built analysis.
The concentrations of cytokines and chemokines were analysed in serum collected from EVAR patients pre-operatively and 6 months post-operatively (
Patient Serum does not Directly Activate Cultured Endothelial Cells.
As the serum levels of some inflammatory cytokines and chemokines were reduced by the EVAR protocol, it was investigated whether these changes would be functionally relevant in an integrated inflammatory model of leukocyte recruitment. Endothelial cells cultured in flow chambers were stimulated with 30% patient serum in endothelial cell culture medium. For comparison, matched endothelial cells were also stimulated with either low (5 U/ml) or high (100 U/ml) dose TNF-α. Unstimulated endothelial cells did not support the adhesion of flowing neutrophils (
Although patient serum did not directly stimulate cultured endothelial cells to recruit flowing neutrophils, it was found that incubation of the endothelial cells with pre-operative serum primed the endothelial cells for responses to TNF-α. Comparing neutrophil adhesion to endothelial cells pre-incubated with different serums prior to activation with 5 U/ml TNF-α, showed that there was a non-significant trend to increased neutrophil recruitment in the presence of patient serum compared to serum from the control cohort (
The Ability of Pre-Operative Sera to Prime Endothelial Cells for Response to TNF-α is Lost when the Biological Activity of IL-1α is Neutralised.
The ability of patient sera to prime endothelial cells was dramatically reduced after EVAR, and this loss of activity was associated with a consistent and significant reduction in the levels of IL1-α and IL-8 in the sera. Thus, it was hypothesised that one of these molecules might be the endothelial cell priming agent. To examine this thesis, a number (n=6) of pre-operative serum samples were re-tested before and after the addition of function neutralising antibodies against IL-8 or IL-1α.
By these studies, IL-1α has been implicated in the molecular and cellular pathology of AAA and is indicated to be a convenient serum biomarker for aneurysm severity and for determining successful outcome of EVAR. It is concluded that EVAR is a procedure which not only prevents AAA rupture, but also reduces levels of chronic systemic inflammation and this can account for the good long term outcome observed in EVAR patients.
Norgren et al. (J. Endovascular Surgery (1997) 4, 169-173) measured levels of TNF-α, IL-6 and IL-8 in EVAR patients pre-operatively, 24 hr post operative and 7 days post-operatively. Levels of each were found to increase following surgical insult, as expected, but returned to baseline by 7 days. Pardoi et al. (J. Endovascular Therapy) measured IL-8 by ELISA in EVAR patients pre-surgery, and up to 72 hrs following surgery, finding that levels increased immediately after surgery, and fell by 72 hrs, although not to pre-operative levels. However, in those studies there was no measurement of IL-1α in the serum of AAA patients. Detection of IL-1α at high concentration in pre-operative serum of AAA patients was a surprising finding contrary to prior indication that IL-1α is not a highly secreted molecule.
