Abstract
Background: The role of hypoxia responsive genes in the pathogenesis of ALS was first suggested when deletions of the hypoxia-responsive element of vascular endothelial growth factor (VEGF) promoter caused a motor neuron disease phenotype in mice. The discovery of ALS-associated mutations in ANG, a hypoxia responsive gene coding for the protein angiogenin, has further supported this pathogenic mechanism in human ALS. In endothelium, angiogenin can regulate expression of VEGF. To date, the patterns of serum angiogenin expression among patients with ALS have not been assessed.
Methods: Serum angiogenin and VEGF levels were quantified at diagnosis in 79 patients with definite or probable ALS and 72 healthy controls, using a quantitative sandwich enzyme-linked immunoassay.
Results: Patients with ALS exhibited higher serum angiogenin (p = 0.006) but not VEGF (p = 0.55) levels than matched control subjects. Subgroup analysis showed a greater elevation in angiogenin levels for spinal- (p < 0.001) than bulbar- (p = 0.11) onset ALS vs controls. At 12 months, angiogenin levels remained elevated. No correlation was noted between angiogenin and VEGF levels (r = −0.08, p = 0.49) in ALS patient serum.
Conclusion: These data suggest a modest elevation in serum angiogenin in ALS at diagnosis. Further investigation will be required to assess the utility of serum angiogenin as a biomarker for ALS and as a predictor of disease progression.
Mice with deletions of the hypoxia-responsive element of vascular endothelial growth factor (VEGF) develop an ALS-like phenotype.1 This observation has led to the hypothesis that hypoxia-responsive proteins may play a role in the pathogenesis of ALS. A higher frequency of at-risk VEGF haplotypes have been associated with lower circulating levels of VEGF2 in ALS patients vs controls. However, further reports concerning serum VEGF expression in ALS have been conflicting. Nygren et al.3 found VEGF levels increased in the serum of affected individuals, whereas other studies have not detected differences in serum VEGF levels.4,5 CSF VEGF concentrations have been elevated in patients after a long duration of spinal ALS, but have been lower in early disease.4,5
Angiogenin, the 14.1-kd product of the hypoxia-responsive gene ANG, has functional similarity to VEGF.6 In endothelial cells, angiogenin stimulates rRNA transcription and is necessary for cell proliferation induced by functionally similar angiogenic proteins including VEGF.6 We have previously reported that possession of a G allele of ANG's rs11701 single nucleotide polymorphism (SNP) confers double the risk of the development of ALS in individuals of Irish or Scottish descent, but not in populations from the United States, England, or Sweden.7,8 This coding SNP is not predicted to affect the known function of angiogenin, although it has not been established whether it alters angiogenin expression. We have also described ALS-associated mutations in ANG, which segregated with disease in one family.8 Although angiogenin is expressed in motor neurons, its exact role in the pathogenesis of ALS remains to be established.
We profiled serum angiogenin and VEGF levels at diagnosis in a large cohort of patients with ALS and investigated their relationship to the known at-risk allele and to clinical phenotypes.
Methods.
Patients.
The study population comprised 79 Irish patients with ALS and 72 age-, sex-, and ethnicity-matched healthy volunteer control subjects. The demographics of the study population are summarized in table 1. All patients fulfilled the El Escorial criteria for definite or probable ALS. Serum was drawn at time of diagnosis, and a follow-up 12-month sample was drawn from 19 ALS patients. Survival statistics were obtained from the Irish ALS register. Survival was defined as the period from first symptom to death. No patient underwent tracheostomy or invasive mechanical ventilation. Subjects were excluded from enrollment if they had a history of inflammatory disease or malignancy or were pregnant. Control subjects had no history of neurologic disease. Informed consent was obtained in all cases, and the study was approved by the Beaumont Hospital Ethics and Medical Research Committee (protocol 49/05).
Table 1 Characteristics of the study population
Quantification of angiogenin and VEGF in serum.
After removal of cells by centrifugation, serum samples were stored at −80 °C until assay. Concentrations of angiogenin and VEGF were measured by ELISA using commercially available kits (Quantikine) purchased from R&D Systems (Abingdon, UK) according to manufacturer's guidelines. All samples were assayed in triplicate. The intra- and interassay coefficients of variation were 3.0% and 6.7% for the angiogenin assay and 4.8% and 4.7% for the VEGF assay.
DNA amplification and genotyping.
Of the patients with ALS included in the present study, 45 had been genotyped for the ANG rs11701 SNP, as previously described.7
Statistical analysis.
Data are shown as mean ± SD. The Shapiro-Wilk test was used to assess the normality of data distribution. Initial comparison between the ALS and control groups was by the independent-samples t test or Mann-Whitney test as appropriate. Because age, sex, and body mass index (BMI) influence serum angiogenin and VEGF levels,9 comparisons were further explored by analysis of covariance (ANCOVA) with those characteristics as covariates. Bonferroni post hoc testing was performed to compare the adjusted levels between subgroups. Logarithmic transformation of the VEGF data was necessary for the multivariate analysis as it was not normally distributed. The correlation analysis was performed using Spearman and Pearson correlation coefficients. The level of significance was set at p < 0.05 in two-tailed tests.
Results.
Serum angiogenin levels.
The overall ALS patient group had higher serum angiogenin levels than controls (p = 0.001) (table 2). Multivariate modeling accounting for the potential influence of age, sex, and BMI over angiogenin levels confirmed the difference between these two groups (F = 7.8, p = 0.006).
Table 2 Serum angiogenin and VEGF levels in ALS patients and controls
When spinal-onset patients, bulbar-onset patients, and controls were considered separately, the analysis of variance gave a difference between the three groups for serum angiogenin (F = 3.6, p = 0.021) (table 2). Male sex was the only covariate found to influence levels (p = 0.035). Bonferroni post hoc comparison between the control group and disease groups revealed an elevation of angiogenin levels for spinal (p < 0.001) but not bulbar (p = 0.11) patients (figure).
Figure. Serum angiogenin and vascular endothelial growth factor (VEGF) levels in patients with ALS vs controls. *Significant difference (p < 0.05) from the control group.
Serum VEGF levels.
No differences in VEGF concentration were observed between the overall ALS and control groups (univariate analysis p = 0.39; multivariate analysis F = 0.36, p = 0.55). Consideration of spinal- and bulbar-onset groups separately did not reveal any differences in serum VEGF levels (intergroup differences: F = 0.36, p = 0.7). Post hoc comparison with controls showed no difference in VEGF levels for spinal (p = 0.5) or bulbar (p = 0.76) patients (figure).
Comparison of baseline levels with follow-up at 12 months.
Follow-up angiogenin and VEGF concentrations at 12 months after diagnosis correlated with initial levels for both peptides (r = 0.6, p = 0.009 for angiogenin; r = 0.8, p < 0.0001 for VEGF). While angiogenin levels did not differ from baseline (371.4 ± 115.7 vs 397.9 ± 81.3 ng/mL, p = 0.55, paired t test), serum VEGF levels were modestly lower at follow-up (200.8 ± 129.8 vs 245.9 ± 154.1 ng/mL, p = 0.047, paired t test).
Influence of serum angiogenin over serum VEGF levels.
No correlation was found between serum angiogenin and VEGF levels either overall (r = 0.06, p = 0.46) or among patients with ALS (r = −0.08, p = 0.49).
Serum angiogenin levels stratified by genotype.
Angiogenin levels were highest among individuals who were homozygous for the at-risk G allele of the rs11701 SNP compared to those who were homozygous for the T allele. However, this did not reach significance (455.4 ± 115.8 vs 376.8 ± 98.7 ng/mL, p = 0.127). No difference was seen for heterozygotes.
Angiogenic growth factors and survival.
To date, definitive survival data are available on 43 of the patients, with mean survival of 32.8 months (± 22.8). There was no correlation noted between survival and serum angiogenin (r = 0.01, p = 0.52) or VEGF levels (r = −0.06, p = 0.69).
Discussion.
We found that serum angiogenin levels are significantly elevated in Irish patients with ALS vs controls. Moreover, the results suggest that the ALS phenotype influences serum angiogenin levels. Angiogenin concentrations were higher in individuals with spinal-onset than with bulbar-onset disease. By contrast, we did not find any differences in serum VEGF between patients with ALS and controls in our population.
Angiogenin was first described in human carcinoma cells,10 and to date, human serum studies have concentrated on its potential role as a marker for tumor progression and for inflammation.11 We have previously characterized a series of ALS-associated ANG mutations, some of which are predicted to reduce angiogenin enzymatic activities.2 As ANG mutations are rare in ALS, here we profiled angiogenin levels independently of mutations. Our findings of elevated serum levels lend support to the hypothesis that angiogenin, like VEGF, may be an important regulatory protein within the neuroaxis. The observed elevation in circulating angiogenin in ALS was modest but reached statistical significance. Moreover, small variations in growth factors may exert a biologic effect through a complex network of molecular interactions.12 The degree of variation observed in our sample would be insufficient for use alone as a diagnostic marker for ALS. The higher levels observed for spinal-onset than for bulbar-onset disease may reflect more widespread involvement of the neuroaxis in spinal ALS. An alternative hypothesis is that the sensitivity of motor neurons to angiogenin may differ along the neuraxis, such that individuals with high baseline angiogenin levels may be protected from a bulbar-onset phenotype. Levels of angiogenin remained stable 12 months on from diagnosis.
Previous reports of serum VEGF in ALS have found elevated levels,3 normal levels,4,5 and low levels.2 These conflicting results may reflect heterogeneous study designs, with levels being measured at different time points in the natural history of the disease. The previous studies may also have been underpowered to detect differences with certainty, as we estimate that a sample size of at least 50 per group is required to detect a 20% difference in levels. It has also recently been suggested that unmatched sex ratios among the four European populations included in the initial VEGF haplotype study may have influenced its findings.13 Our observations are best compared to those of Devos et al.,4 who restricted inclusion to early ALS. In our larger population of patients newly diagnosed with ALS, we found no alteration of serum VEGF expression, confirming and extending the data suggesting that VEGF expression is not upregulated in the early stages of the disease.
No correlation was observed in our study between serum levels of angiogenin and VEGF. In endothelial cells, angiogenin-induced ribosome biosynthesis has been suggested to act as a necessary step for cell proliferation by VEGF.6 Downregulation of angiogenin by small interfering RNA (siRNA) and antisense oligonucleotides attenuates the angiogenic activity of VEGF in cell culture.6 In serum, however, a large cohort study9 among 947 healthy individuals demonstrated no correlation of angiogenin levels with VEGF. Our findings extend this observation to a disease cohort with increased serum angiogenin. One possible explanation is that angiogenin signaling leads to a different profile of responses in the serum compared to endothelium.
It remains uncertain whether the at-risk G allele of the rs11701 SNP reduces angiogenin function. If the risk exerted by the G allele is dose dependent in the Irish population, it follows that individuals homozygote for the G allele would show the highest angiogenin serum levels. In our patients, the trend toward elevated angiogenin levels observed among G allele homozygotes did not reach significance (p = 0.11).
We did not identify a relationship between angiogenin levels and prognosis. However, our current data are biased toward a group with shorter survival than usually seen in our population.14 We have noted that the greatest elevation in angiogenin was seen in limb-onset disease among a group who are known to exhibit longer survival.15 Further work is required to determine the biologic function of angiogenin within the neuroaxis and to establish whether serum levels can be used as a prognostic indicator in ALS.
Acknowledgment
The authors thank the patients, their families, and the referring neurologists for their participation in this study.
Footnotes
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Supported by a translational research grant from the Charitable Infirmary Charitable Trust (S.C.) and by the Health Research Board of Ireland (M.G., O.H.).
Disclosure: Royal College of Surgeons in Ireland hold a patent on angiogenin for therapeutic uses in ALS. The College asserts its rights over this patent and all proceeds generated thereof for its charitable aims (Registered Charity No. CHY 127). The authors M.J.G. and O.H. are listed as inventors on this patent.
Received May 31, 2006. Accepted in final form July 28, 2006.
References
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