Daniel T. Laskowitz

Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury

IntroductionAcute kidney injury (AKI) can evolve quickly and clinical measures of function often fail to detect AKI at a time when interventions are likely to provide benefit. Identifying early markers of kidney damage has been difficult due to the complex nature of human AKI, in which multiple etiologies exist. The objective of this study was to identify and validate novel biomarkers of AKI.MethodsWe performed two multicenter observational studies in critically ill patients at risk for AKI - discovery and validation. The top two markers from discovery were validated in a second study (Sapphire) and compared to a number of previously described biomarkers. In the discovery phase, we enrolled 522 adults in three distinct cohorts including patients with sepsis, shock, major surgery, and trauma and examined over 300 markers. In the Sapphire validation study, we enrolled 744 adult subjects with critical illness and without evidence of AKI at enrollment; the final analysis cohort was a heterogeneous sample of 728 critically ill patients. The primary endpoint was moderate to severe AKI (KDIGO stage 2 to 3) within 12 hours of sample collection.ResultsModerate to severe AKI occurred in 14% of Sapphire subjects. The two top biomarkers from discovery were validated. Urine insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, a key mechanism implicated in AKI, together demonstrated an AUC of 0.80 (0.76 and 0.79 alone). Urine [TIMP-2]·[IGFBP7] was significantly superior to all previously described markers of AKI (P <0.002), none of which achieved an AUC >0.72. Furthermore, [TIMP-2]·[IGFBP7] significantly improved risk stratification when added to a nine-variable clinical model when analyzed using Cox proportional hazards model, generalized estimating equation, integrated discrimination improvement or net reclassification improvement. Finally, in sensitivity analyses [TIMP-2]·[IGFBP7] remained significant and superior to all other markers regardless of changes in reference creatinine method.ConclusionsTwo novel markers for AKI have been identified and validated in independent multicenter cohorts. Both markers are superior to existing markers, provide additional information over clinical variables and add mechanistic insight into AKI.Trial registrationClinicalTrials.gov number NCT01209169.

Simvastatin Reduces Vasospasm After Aneurysmal Subarachnoid Hemorrhage: Results of a Pilot Randomized Clinical Trial

Background and Purpose— Cerebral vasospasm remains a major source of morbidity after aneurysmal subarachnoid hemorrhage (SAH). We demonstrate that simvastatin reduces serum markers of brain injury and attenuates vasospasm after SAH. Methods— Patients with angiographically documented aneurysmal SAH were randomized within 48 hours of symptom onset to receive either simvastatin (80 mg daily; n=19) or placebo (n=20) for 14 days. Plasma alanine aminotransferase, aspartate aminotransferase, and creatine phosphokinase were recorded weekly to evaluate laboratory evidence of hepatitis or myositis. Serum markers of brain injury were recorded daily. The primary end point of vasospasm was defined as clinical impression (delayed ischemic deficit not associated with rebleed, infection, or hydrocephalus) in the presence of ≥1 confirmatory radiographic test (angiography or transcranial Doppler demonstrating mean VMCA >160 m/sec). Results— There were no significant differences in laboratory-defined transaminitis or myositis between groups. No patients developed clinical symptoms of myopathy or hepatitis. Plasma von Willebrand factor and S100&bgr; were decreased 3 to 10 days after SAH (P<0.05) in patients receiving simvastatin versus placebo. Highest mean middle cerebral artery transcranial Doppler velocities were significantly lower in the simvastatin-treated group (103±41 versus 149±47; P<0.01). In addition, vasospasm was significantly reduced (P<0.05) in the simvastatin-treated group (5 of 19) compared with those who received placebo (12 of 20). Conclusion— The use of simvastatin as prophylaxis against delayed cerebral ischemia after aneurysmal SAH is a safe and well-tolerated intervention. Its use attenuates serum markers associated with brain injury and decreases the incidence of radiographic vasospasm and delayed ischemic deficit.

Central nervous system injury associated with cardiac surgery

Millions of individuals with coronary artery or valvular heart disease have been given a new chance at life by heart surgery, but the potential for neurological injury is an Achilles heel. Technological advancements and innovations in surgical and anaesthetic technique have allowed us to offer surgical treatment to patients at the extremes of age and infirmity-the group at greatest risk for neurological injury. Neurocognitive dysfunction is a complication of cardiac surgery that can restrict the improved quality of life that patients usually experience after heart surgery. With a broader understanding of the frequency and effects of neurological injury from cardiac surgery and its implications for patients in both the short term and the long term, we should be able to give personalised treatments and thus preserve both their quantity and quality of life. We describe these issues and the controversies that merit continued investigation.

Simvastatin Increases Endothelial Nitric Oxide Synthase and Ameliorates Cerebral Vasospasm Resulting From Subarachnoid Hemorrhage

Background and Purpose— Endothelial nitric oxide synthase (eNOS) activity is decreased after subarachnoid hemorrhage (SAH). Simvastatin increases eNOS activity. We hypothesized that simvastatin would increase eNOS protein and ameliorate SAH-induced cerebral vasospasm. Methods— Mice were treated with subcutaneous simvastatin or vehicle for 14 days and then subjected to endovascular perforation of the right anterior cerebral artery or sham surgery. Three days later, neurological deficits were scored (5 to 27; 27=normal), and middle cerebral artery diameter and eNOS protein were measured. The study was repeated, but simvastatin treatment was started after SAH or sham surgery. Results— In SAH mice, simvastatin pretreatment increased middle cerebral artery diameter (SAH-simvastatin=74±22 &mgr;m, SAH-vehicle=52±18 &mgr;m, P =0.03; sham-simvastatin=102±8 &mgr;m, sham-vehicle=105±6 &mgr;m). Pretreatment reduced neurological deficits (SAH-simvastatin=25±2, SAH-vehicle=20±2, P =0.005; sham-simvastatin and sham-vehicle=27±0). Simvastatin pretreatment also increased eNOS protein. Simvastatin posttreatment caused a modest increase in middle cerebral artery diameter in SAH mice (SAH-simvastatin=56±12 &mgr;m, SAH-vehicle=45±4 &mgr;m, P =0.03; sham-simvastatin=92±13 &mgr;m, sham-vehicle=99±10 &mgr;m) and reduced neurological deficits (SAH-simvastatin=21±1, SAH-vehicle=19±2, P =0.009). Simvastatin posttreatment did not significantly increase eNOS protein. Conclusions— Simvastatin treatment before or after SAH attenuated cerebral vasospasm and neurological deficits in mice. The mechanism may be attributable in part to eNOS upregulation.

Novel Diagnostic Test for Acute Stroke

Background and Purpose— The absence of a widely available and sensitive diagnostic test for acute cerebral ischemia remains a significant limitation in the diagnosis and management of stroke. The objective of this study was to examine the feasibility of developing a diagnostic panel of blood-borne biochemical markers of cerebral ischemia. Methods— Serial blood samples were obtained from patients (n=65 with suspected ischemic stroke, n=157 control subjects) presenting to an academic medical center emergency department. We analyzed 26 blood-borne markers believed to play a role in the ischemic cascade and created a 3-variable logistic regression model to predict the clinical diagnosis of stroke, defined as persistent neurological symptoms of cerebral ischemia lasting >24 hours. Results— Of the 26 blood-borne markers analyzed, univariate logistic analysis revealed that 4 were highly correlated with stroke (P <0.001): a marker of glial activation (S100&bgr;), 2 markers of inflammation (matrix metalloproteinase-9 and vascular cell adhesion molecule), and 1 marker of thrombosis (von Willebrand factor). When the outcome level was set to a cutoff of P =0.1, our logistic model provided a sensitivity and specificity of 90% for predicting stroke. Conclusions— A panel of blood-borne biochemical markers may be helpful in identifying patients with acute cerebral ischemia who could benefit from urgent care. Such a test may also be helpful in identifying stroke patients in the prehospital setting so that they could be fast-tracked to an institution equipped to care for patients with acute stroke.

Apolipoprotein E and the CNS response to injury.

Apolipoprotein E (apoE) is a multifunctional protein with an expanding role in the neurobiology of disease. Although originally described in the context of cholesterol metabolism, interest in the neurobiology of apoE has intensified following the association between apoE genotype and risk of developing Alzheimers disease. Recent clinical observations also suggest that apoE genotype may influence recovery after a variety of neurological insults. Thus, in addition to the study of disease-specific mechanisms by which apoE may modulate susceptibility of developing Alzheimers disease, there has been an increasing focus on its role in modulating the CNS response to acute injury. Although the neurobiology of apoE in the injured brain remains incompletely defined, there is evidence to suggest neurotrophic, immunomodulatory, and antioxidant effects.

Apolipoprotein E-Deficient Mice Have Increased Susceptibility To Focal Cerebral Ischemia

Recent evidence suggests that apolipoprotein E (ApoE) plays a role in neurologic disease. This experiment compared the neurologic and histologic outcome of ApoE-deficient mutant and wild-type mice subjected to a 60- or 90-minute episode of middle cerebral artery filament occlusion and a recovery interval of 24 hours. With 60 minutes of ischemia, there was no mortality. Apolipoprotein E-deficient mice had larger infarcts (cortex: ApoE deficient = 20 mm3 ± 12, wild-type = 9 ± 7 mm3, P = 0.03; subcortex: ApoE deficient = 22 ± 7 mm3, wild-type = 16 ± 7 mm3, P = 0.07). Hemiparesis was less severe in wild-type animals (P = 0.02). After 90 minutes of ischemia, mortality in ApoE-deficient mice (n = 10) was 40% versus 0% in wild-type mice (n = 10; P = 0.09). Intraparenchymal hemorrhage was found in 3 of the 4 dead mice. No difference in cortical (ApoE deficient = 37 ± 8 mm3; wild-type = 31 ± 18 mm3; P = 0.49) or subcortical (ApoE deficient = 30 ± 11 mm3; wild-type = 32 ± 18 mm3; P = 0.78) infarct volumes was present among survivors. ApoE-deficient mice had a prolonged activated partial thromboplastin time and increased fibrinogen concentration. This data supports the hypothesis that apolipoprotein E plays a role in the pathophysiology of ischemic brain damage.

Early Biomarkers of Stroke

BACKGROUND The diagnosis and management of acute ischemic stroke are limited by the lack of rapid diagnostic assays for use in an emergency setting. Computed tomography (CT) scanning is used to diagnose hemorrhagic stroke but is relatively ineffective (<33% sensitive) in detecting ischemic stroke. The ability to correlate blood-borne protein biomarkers with stroke phenotypes would aid in the development of such rapid tests. METHODS ELISAs for >50 protein biomarkers were developed for use on a high-throughput robotic workstation. These assays were used to screen plasma samples from 214 healthy donors and 223 patients diagnosed with stroke, including 82 patients diagnosed with acute ischemic stroke. Marker assay values were first compared by univariate analysis, and then the top markers were subjected to multivariate analysis to derive a marker panel algorithm for the prediction of stroke. RESULTS The top markers from this analysis were S-100b (a marker of astrocytic activation), B-type neurotrophic growth factor, von Willebrand factor, matrix metalloproteinase-9, and monocyte chemotactic protein-1. In a panel algorithm in which three or more marker values above their respective cutoffs were scored as positive, these five markers provided a sensitivity of 92% at 93% specificity for ischemic stroke samples taken within 6 h from symptom onset. CONCLUSION A marker panel approach to the diagnosis of stroke may provide a useful adjunct to CT scanning in the emergency setting.

Apolipoprotein E suppresses glial cell secretion of TNFα

Apolipoprotein E (apoE) is a 299 amino acid protein with multiple biological functions. Initially described in the context of cholesterol metabolism, apoE also has immunomodulatory properties and recent evidence has implicated a role for apoE in neurological disease. One possibility is that apoE, which is the predominant apolipoprotein produced intra-axially, may modify the CNS response to acute and chronic injury. We prepared mixed neuronal-glial cultures from apoE deficient mouse pups and measured secretion of TNF alpha after stimulation with lipopolysaccharide (LPS) in the presence and absence of human recombinant apoE3 and E4. We demonstrate that preincubation with apoE blocks glial secretion of TNF alpha in a dose-dependent manner. This effect is independent of any direct effect of apoE on cell viability and is greatest when apoE is preincubated with the cell culture for 24 h.

Downregulation of Microglial Activation by Apolipoprotein E and ApoE-Mimetic Peptides

Apolipoprotein E plays an important role in recovery from acute brain injury and risk of developing Alzheimers disease. We demonstrate that biologically relevant concentrations of apoE suppress microglial activation and release of TNFalpha and NO in a dose-dependent fashion. Peptides derived from the apoE receptor-binding region mimic the effects of the intact protein, whereas deletion of apoE residues 146-149 abolishes peptide bioactivity. These results are consistent with the hypothesis that apoE modulates microglial function by binding specific cell surface receptors and that the immunomodulatory effects of apoE in the central nervous system may account for its role in acute and chronic neurological disease.