The aim of this study was to investigate the relationship between ADMA and SDMA as mediators of oxidative stress and endothelial dysfunction on the one hand and the inflammatory response on the other hand in patients with acute ischemic stroke.
After the acute event of stroke, the proinflammatory cytokine IL-6 is rapidly released by activated cells in brain tissue and peripheral blood . In contrast to this, SDMA and ADMA levels increase during the first day. SDMA and ADMA are not correlated with IL-6 during the first 24 hours after stroke onset but we observed a correlation days after the event. Also in patients with coronary artery disease and chronic kidney disease, SDMA and IL-6 levels were correlated [19, 20]. Recently Tripepi et al. showed that increased levels of ADMA in combination with high IL-6 and CRP levels are predictive for death and cardiovascular events in patients with end-stage renal disease . Cell culture experiments suggest a direct link between dimethylarginines and IL-6. SDMA has been shown to increase expression of IL-6 in monocytes, whereas in adipocytes ADMA triggers the expression of IL-6 via activation of nuclear factor-kappa B . However, since levels of dimethylarginines peak when IL-6 has already significantly decreased during the first day after stroke, ADMA and SDMA are unlikely to induce the hyperacute increase of IL-6 in the first hours after stroke. Of note, in healthy young adults an inverse correlation of IL-6 with DDAH2 expression has been shown . Decreased DDAH2 expression leads to an accumulation of ADMA in the human body. Thus in case of stroke, the relationship between IL-6 and ADMA is contrary to the observations in cell cultures. ADMA might increase at least in part as a response to high IL-6 levels.
In the current study, SDMA plasma levels were correlated with MCP-1 at the early stage but also days after the acute event independently of renal function. The chemokine MCP-1 recruits monocytes/macrophages to the injury site after stroke onset, which triggers the inflammatory reaction by further release of mediators . Moreover, in vitro data suggest that SDMA triggers inflammation by enhancing reactive oxygen species (ROS) production of monocytes via modulation of store-operated calcium channels . In contrast to SDMA, ADMA was not correlated to MCP-1 in this study, although ADMA has been shown to increase MCP-1 in vitro.
Considering data from the literature, both ADMA and SDMA levels are expected to increase in the state of chronic inflammation [27, 28] and to decrease in the state of acute inflammation. Zoccali et al. showed in 17 patients with bacterial infection that during the acute phase, patients displayed very high levels of CRP, IL-6, procalcitonin and nitrotyrosine . When infection resolved, ADMA levels rose significantly while SDMA levels remained unmodified. After kidney donation of healthy subjects, ADMA levels temporarily decreased in the state of acute elevated inflammation . Decline of the inflammation markers (IL-6 and CRP) was accompanied with an increase in ADMA levels. Further, Blackwell et al. showed that ADMA decreased rapidly during the first 48 hours of acute inflammation after total knee arthroplasty .
One explanation for the difference between stroke and other types of acute systemic inflammation might be the fact that both oxidative stress and inflammation are induced by the detrimental cascade after acute ischemic stroke. This may not prove the mechanistical association but at least that levels are increased in parallel. The missing correlation between dimethylarginines and the marker of the extent of brain cell damage S100B in the current study suggests that the increase of dimethylarginines is not primarily caused by the amount of degradation of malperfused brain tissue. Moreover, experimental data suggest that a link between ADMA levels and infarct size is lacking. Leypoldt et al. investigated the infarct size in a transient middle cerebral artery occlusion (tMCAO) model of human DDAH-1 (hDDAH-1) transgenic (TG) mice . Size of infarction did not differ between hDDAH-1 TG mice and wild-type (WT) littermates. But also DDAH activity in the brain and cerebral ADMA tissue levels in TG mice remained unchanged. However, there may be differences between animal models and patients, since in a rat model of tMCAO ADMA levels in serum and brain were unchanged, while they were significantly decreased in cerebrospinal fluid (CSF) .
ADMA might be involved in post-ischemic recovery, as ADMA and TIMP-1 were independently correlated during the first hours and also days after the event. TIMP-1 is an endogenous MMP-9 inhibitor. Glial cell expression of TIMP-1 has been shown to be regulated by microglia . TIMP-1 concentrations are elevated early after ischemic stroke onset . In monocytes of 25 ischemic stroke patients, TIMP-1 expression was increased during the first days after the event . Elevated levels of TIMP-1 might limit matrix proteolysis and thereby play a role in tissue remodelling . Moreover, a possible involvement of ADMA in post-ischemic recovery is supported by the fact that ADMA was inversely correlated with MMP-9 at 12 hours after stroke onset in our study. MMP-9 is known as a member of the zinc-binding proteolytic enzymes, which degrades extracellular matrix (ECM) proteins found in the basal lamina of blood vessels. Thereby, increased MMP-9 levels are associated with haemorrhagic transformation after ischemic stroke . In a mouse model of ischemic stroke, the nonselective NOS inhibitor N-omega-nitro-L-arginine (L-NA) decreased MMP-9 expression . In a recently published study in rats, lowering of NO by the NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) levels led to reduced blood–brain barrier disturbance . As a nonselective NOS inhibitor, ADMA might have similar effects, though we do not have direct evidence yet. Remarkably, MMP-9 levels were not elevated in stroke patients compared with those in controls in our study. At day 3, levels of MMP-9 in patients were even significantly lower. While several studies showed increased MMP-9 levels in stroke patients compared to controls [41–43], few studies did not detect clear differences [44, 45]. In accordance to our results, in two studies median levels of MMP-9 were - although not significantly - lower in stroke patients than in controls at 3 days and 2 to 5 days after stroke, respectively [45, 46]. Further studies need to clarify these controversial findings.
In addition to the shown association of dimethylarginines and inflammation, we cannot exclude an effect of pharmacological treatment on dimethylarginine levels in our patients. Some drugs lower dimethylarginine levels, such as statins, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers [47, 48]. Metabolic changes like impaired glucose tolerance and stroke-associated neurohumoral changes might also have an effect on dimethylarginine levels. ADMA has repeatedly been shown to increase with high glucose levels , insulin resistance  and hyperthyroidism . Although our analysis did not show an association between pharmacological treatment, metabolic changes and dimethylarginine levels, we cannot rule out an effect of these factors on the plasma levels of ADMA and SDMA since the patient cohort was too small for such an analysis. Furthermore, our data did not show differences in levels of dimethylarginines in regard to stroke etiology. This is in accordance with data reported by Brouns et al. who investigated ADMA levels in the CSF of patients with stroke . In contrast, Scherbakov et al. found plasma concentrations of ADMA elevated predominantly in patients with atherothrombotic and cardioembolic stroke and not in those with lacunar infarction or undetermined cause of stroke . In Swedish stroke patients, serum concentrations of ADMA were increased in cardioembolic stroke, but not in noncardioembolic stroke . So far these differences have not been explained pathophysiologically. However, these controversial data may result from different patient populations.
Of note, in the present study SDMA levels were significantly higher in controls than in stroke patients at 6 hours. The concentration of SDMA largely depends on renal function as it is mostly excreted by the kidney. Therefore SDMA is a sensitive marker for slight renal impairment. Higher SDMA levels in controls suggested lower eGFR in this group. Comparison of the respective data, however, showed no significant difference. As expected SDMA concentrations in the patients were inversely correlated with eGFR at baseline at each time point. Similar results have been observed in other studies [29, 52]. It shall be emphasised, however, that according to the results of the multivariate regression analysis which included eGFR into the statistical model, the observed association between concentrations of SDMA and inflammatory markers in this study is independent of renal function.
Our data indicate for the first time an association between dimethylarginines as mediators of oxidative stress and inflammation after ischemic stroke. This link points to a potential role of dimethylarginines in the detrimental cascade in stroke pathophysiology and might partly explain the correlation of dimethylarginines with stroke outcome [6, 53].
However, our study has some limitations. It remains unclear whether elevated dimethylarginine levels are the cause or the result of increased inflammation, which deserves further investigation in experimental models. Furthermore, the number of patients included into this study was small. However, since we collected serial blood samples in short intervals after ischemic stroke we were able to describe the temporal change of levels of dimethylarginines and mediators of inflammation during the first days after the event, which has never been done before. Finally, only few severe strokes were included in this study. Since inflammation and oxidative stress strongly increase in severe strokes, inclusion of a large number of mild to moderate strokes in this study might have led to underestimation of the association between dimethylarginines and inflammatory mediators.