Low levels of the vascular endothelial growth factor in CSF from early ALS patients

Patients and methods.

Serum, plasma, and CSF from ALS and control subjects were obtained with informed consent. Subject characteristics (24 sporadic ALS patients and 34 control subjects) are summarized in the table⇓. None of the subjects had major hypoxemia or pulmonary disease, although five ALS patients showed slight hypoxemia (P_ao₂: 73 mm Hg). At the time of lumbar puncture, 9 patients fulfilled the El Escorial diagnostic criteria for definite ALS, 13 for probable ALS, and 2 for possible ALS. After a 1-year follow-up, 16 patients had definite and 8 probable ALS. Normal control CSF and serum were obtained from 19 patients with suspected neurologic disease but classified as negative after testing; final diagnoses included headache without meningeal hemorrhage and a range of psychological disorders. Neurologic control CSF was obtained from 15 patients for whom the diagnosis included hydrocephaly, Wernicke syndrome, noninflammatory neuropathy, Parkinson disease, dementia, supranuclear palsy, cervical spondylotic myelopathy, vascular encephalopathy, spinal cord ischemia, oculomotor palsy, and idiopathic late-onset cerebellar ataxia. In light of the abnormal activation of HIF by cytokines,4 we chose not to include inflammatory diseases. Only cytologically and biochemically normal CSFs were analyzed.

CSF and postcentrifugation blood sample supernatants were stored at −80 °C. VEGF levels were determined using quantitative sandwich enzyme immunoassay techniques, that is, a chemiluminescent assay (QuantiGlo; R&D Systems, Wiesbaden, Germany) for undiluted CSF and an ELISA (Quantikine; R&D Systems) for serum and plasma analyses (intra-assay and interassay precision <10%). These assays measure unbound recombinant and natural human VEGF₁₆₅. All VEGF measurements were carried out on the same day with the same kit.

Statistical analysis included analysis of variance on ranks (similar to a Kruskal-Wallis test) with the Bonferroni posthoc test (a conservative test used when there are few comparisons between groups), the χ² test, and Spearman rank correlation coefficient.

Results.

Electrophoresis analysis results did not significantly differ between the groups (see the table⇑), whereas CSF VEGF levels did (F = 8.9, p = 0.0001), with lower levels in ALS patients than in the neurologic and normal control subjects. There were no significant differences between the neurologic and normal control subjects (see the table⇑; figure, A). In all three groups, VEGF plasma levels were significantly lower than serum levels (z = −3.2, p = 0.001) and significantly correlated (r = 0.7, p = 0.0001). Plasma and serum VEGF levels were significantly different for the three groups (F = 7.8, p = 0.001; F = 5.1, p = 0.002, respectively), with higher levels in neurologic control subjects than in ALS patients and normal control subjects. There were no significant differences between ALS patients and normal control subjects (see the table⇑ and figure, B). The clinical presentation of the ALS patients displaying the highest and the lowest VEGF concentrations did not differ. No differences in VEGF level were observed when comparing hypoxemic and normoxemic ALS subgroups. No significant association was found between CSF VEGF levels and the onset form of ALS. VEGF CSF levels did not correlate linearly with age, plasma, or serum levels.

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Figure. (A) CSF levels of vascular endothelial growth factor (VEGF) in ALS patients, neurologic control subjects, and normal control subjects. Median values and first and third quartiles. *Significant difference (p < 0.05) between the given condition and the two others. (B) Serum levels of VEGF in ALS patients, neurologic control subjects, and normal control subjects. Median values and first and third quartiles. *Significant difference (p < 0.05) between the given condition and the two others.

Discussion.

We found significantly lower baseline CSF VEGF levels in ALS patients vs control subjects during the early phase of the disease, suggesting the potential involvement of VEGF₁₆₅ in ALS.1,3⇓ VEGF promoter polymorphisms are found only in some ALS patients.3 Thus, the low CSF VEGF levels found in most of our ALS patients suggest the involvement of other mechanisms lowering VEGF production. The latter is notably influenced by a range of cytokines,4 and VEGF expression appears to be regulated through dual, interdependent mechanisms involving HIF-1 (directly and indirectly) via different transcription factors, particularly nuclear factor-κB-mediated cyclo-oxygenase-2 expression and prostaglandin E₂ (PGE₂).5 In particular, abnormally high PGE₂ levels have been demonstrated in ALS patients.6 Low CSF levels could lead to decreased VEGF₁₆₅-dependent neuroprotection, during either chronic reduced neural vascular perfusion or repetitive hypoxic episodes. This would finally result in selective motor neuron degeneration,2 combined with oxidative damage.7

A previous study failed to detect VEGF in CSF, possibly owing to the low sensitivity (5 pg/mL) of the ELISA kits used.8 Here, using a more sensitive immunoassay (1.76-pg/mL detection limit), we were able to detect VEGF in CSF for the first time. Furthermore, this study did not find evidence for a significant decrease in VEGF levels in human postmortem spinal cord samples from 13 sporadic ALS patients.8 However, this tissue may not be relevant to the initial pathologic process. Equally, the mean range of VEGF levels in the ventral horn was lower in ALS, but this difference was not significant, probably because of the small sample size and the high intervariability.8

Significantly higher VEGF levels were also demonstrated in sera from patients with advanced ALS; this was supposedly related to excessive collateral sprouting of the reinnervation process in chronic skeletal muscle ischemia.8 This hypothesis could explain why we did not find significant increased serum levels of VEGF in patients with early ALS who had not yet displayed diffuse amyotrophy. As previously demonstrated, we found that plasma levels were significantly lower than sera levels, owing to the release of platelet-derived VEGF during coagulation.9 We therefore recommended using plasma rather than serum. In contrast to a recent genetic study,3 VEGF plasma levels were not lower in ALS patients than in controls, probably because we did not perform haplotype selection in our smaller sample of subjects. Our findings suggest 1) that VEGF expression may be differently regulated in CSF and plasma, 2) that CSF is more sensitive than plasma to pathologic changes in VEGF, and 3) that baseline VEGF plasma levels might not be relevant in early ALS. However, we also found significantly higher levels of VEGF plasma levels in neurologic control subjects, possible representing either a consequence of amyotrophy8 or a compensatory mechanism. Indeed, one might ask whether the physiopathologic dynamics of VEGF would also involve a lack of up-regulation in response to hypoxia, as suggested both previously1,3⇓ and here by the lack of high VEGF levels in our five hypoxic ALS patients. It will be necessary to perform additional in-depth studies (including VEGF polymorphism analyses) on a larger population to determine whether the possible lack of up-regulation in ALS involves disturbed expression of the hypoxia-inducible, neurotrophic factors involved in VEGF-dependent neuroprotection.