Michael Knoflach

Recanalization after thrombolysis in stroke patients Predictors and prognostic implications

Objective: To estimate rates, predictors, and prognostic importance of recanalization in an unselected series of patients with stroke treated with IV thrombolysis. Methods: We performed a CT angiography or transcranial Doppler (TCD) follow-up examination 24 hours after IV thrombolysis in 64 patients with documented occlusion of the intracranial internal carotid or middle cerebral artery (MCA). Complete recanalization was defined by a rating of 3 on the Thrombolysis in Myocardial Infarction or 4/5 on the Thrombolysis in Brain Ischemia grading scales. Information about risk factors, clinical features, and outcome was prospectively collected by standardized procedures. Results: Complete recanalization was achieved in 36 of the 64 patients (56.3%). There was a nonsignificant trend of recanalization rates to decline with a more proximal site of occlusion: 68.4% (M2 segment of MCA), 53.1% (M1 segment), and 46.2% (carotid T) (p for trend = 0.28). Frequencies of vessel reopening were markedly reduced in subjects with diabetes (9.1% vs 66.0% in nondiabetics, p < 0.001) and less so in subjects with additional extracranial carotid occlusion (p = 0.03). Finally, complete recanalization predicted a favorable stroke outcome at day 90 independently of the information provided by age, NIH Stroke Scale, and onset-to-needle time. Conclusions: We found a high rate of vessel recanalization after IV thrombolysis occlusion. However, recanalization was infrequent in patients with diabetes and extracranial carotid occlusion. Information on recanalization was a powerful, early predictor for clinical outcome.

Cardiovascular Risk Factors and Atherosclerosis in Young Males: ARMY Study (Atherosclerosis Risk-Factors in Male Youngsters)

Background—Necropsy studies suggest that atherosclerosis begins in childhood, but in vivo confirmation of this concept is sparse and limited to selected population samples. Furthermore, new risk concepts of atherosclerosis focusing on inflammation, infections, and immunity have not yet been evaluated in this age group. Methods and Results—This study was conducted in a sample of 141 17- to 18-year-old white males homogenous in age and sex. In addition to classic risk factors, C-reactive protein and the humoral and cellular immune reactivity to heat-shock proteins (HSPs) were assessed. Intima-media thickness (IMT) was quantified at 4 vessel segments of the carotid and femoral arteries. High IMT was considered to be present if the IMT of at least 1 vessel segment exceeded the 90th percentile. In a multivariate logistic regression analysis, cigarette smoking, high diastolic blood pressure, prominent immune reactivity to human and/or mycobacterial HSP60s, alcohol consumption (inverse), and low HDL cholesterol levels were all associated with high IMT. The prevalence of high IMT substantially increased from 0 to 60% when the number of risk conditions in a single individual increased from 0 to 4 (P <0.001 for linear trend). Conclusions—Our study supports the concept that atherosclerosis begins in the first decades of life and suggests a role of the immune system, especially immunoreactivity against HSP60s, in atherosclerosis of young individuals.

The osteoprotegerin/RANK/RANKL system: a bone key to vascular disease

Owing to the common coincidence of osteoporosis and vascular disease, pathophysiological links between both disorders have long been sought. The osteoprotegerin (OPG)/receptor activator of NF-κB (RANK)/receptor activator of NF-κB ligand (RANKL) cytokine network, a key regulatory system in bone homeostasis, has been implicated recently in vascular calcification, changes in matrix composition and diabetic macroangiopathy, aortic aneurysm development, heart failure and, most importantly, advanced atherosclerosis, plaque destabilization and manifestation of cardiovascular diseases. The concept of an active role of RANKL and OPG in vascular pathophysiology is intriguing and is gaining increasing support from both epidemiological and basic research. OPG serum level is considered to be a stable and reliable indicator of the overall activity of the OPG/RANK/RANKL axis and may find application as a biomarker of vascular risk and prognosis. RANKL in turn may be a suitable target for novel therapies. Pharmacological strategies for specific interference with the OPG/RANK/RANKL axis are currently being developed and evaluated in osteoporosis therapy.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59±2 pN, which correlated nicely to the 51±1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Cadmium Is a Novel and Independent Risk Factor for Early Atherosclerosis Mechanisms and In Vivo Relevance

Objectives—Although cadmium (Cd) is an important and common environmental pollutant and has been linked to cardiovascular diseases, little is known about its effects in initial stages of atherosclerosis. Methods and Results—In the 195 young healthy women of the Atherosclerosis Risk Factors in Female Youngsters (ARFY) study, cadmium (Cd) level was independently associated with early atherosclerotic vessel wall thickening (intima-media thickness exceeding the 90th percentile of the distribution; multivariable OR 1.6[1.1.–2.3], P=0.016). In line, Cd-fed ApoE knockout mice yielded a significantly increased aortic plaque surface compared to controls (9.5 versus 26.0 mm2, P<0.004). In vitro results indicate that physiological doses of Cd increase vascular endothelial permeability up to 6-fold by (1) inhibition of endothelial cell proliferation, and (2) induction of a caspase-independent but Bcl-xL-inhibitable form of cell death more than 72 hours after Cd addition. Both phenomena are preceded by Cd-induced DNA strand breaks and a cellular DNA damage response. Zinc showed a potent protective effect against deleterious effects of Cd both in the in vitro and human studies. Conclusion—Our research suggests Cd has promoting effects on early human and murine atherosclerosis, which were partly offset by high Zn concentrations.

Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus.

Hepatic insulin resistance is a driving force in the pathogenesis of type 2 diabetes mellitus (T2DM) and is tightly coupled with excessive storage of fat and the ensuing inflammation within the liver. There is compelling evidence that activation of the transcription factor nuclear factor-κB (NF-κB) and downstream inflammatory signaling pathways systemically and in the liver are key events in the etiology of hepatic insulin resistance and β-cell dysfunction, although the molecular mechanisms involved are incompletely understood. We here test the hypothesis that receptor activator of NF-κB ligand (RANKL), a prototypic activator of NF-κB, contributes to this process using both an epidemiological and experimental approach. In the prospective population-based Bruneck Study, a high serum concentration of soluble RANKL emerged as a significant (P < 0.001) and independent risk predictor of T2DM manifestation. In close agreement, systemic or hepatic blockage of RANKL signaling in genetic and nutritional mouse models of T2DM resulted in a marked improvement of hepatic insulin sensitivity and amelioration or even normalization of plasma glucose concentrations and glucose tolerance. Overall, this study provides evidence for a role of RANKL signaling in the pathogenesis of T2DM. If so, translation to the clinic may be feasible given current pharmacological strategies to lower RANKL activity to treat osteoporosis.

Functional recovery after ischemic stroke—A matter of age Data from the Austrian Stroke Unit Registry

Objective: To analyze the association between patient age and good functional outcome after ischemic stroke with special focus on young patients who were numerically underrepresented in previous evaluations. Methods: Of 43,163 ischemic stroke patients prospectively enrolled in the Austrian Stroke Unit Registry, 6,084 (14.1%) were ≤55 years old. Functional outcome was available in a representative subsample of 14,256 patients free of prestroke disability, 2,223 of whom were 55 years or younger. Herein we analyzed the effects of age on good functional outcome 3 months after stroke (modified Rankin Scale score ≤2). Results: Good outcome was achieved in 88.2% (unadjusted probability) of young stroke patients (≤55 years). In multivariable analysis, age emerged as a significant predictor of outcome independent of stroke severity, etiology, performance of thrombolysis, sex, risk factors, and stroke complications. When the age stratum 56–65 years was used as a reference, odds ratios (95% confidence interval [95%CI]) of good outcome were 3.4 (1.9–6.4), 2.2 (1.6–3.2), and 1.5 (1.2–1.9) for patients aged 18–35, 36–45, and 46–55 years and 0.70 (0.60–0.81), 0.32 (0.28–0.37), and 0.18 (0.14–0.22) for those aged 66–75, 76–85, and >85 years (p < 0.001). In absolute terms, the regression-adjusted probability of good outcome was highest in the age group 18–35 years and gradually declined by 3.1%–4.2% per decade until age 75 with a steep drop thereafter. Findings applied equally to sexes and patients with and without IV thrombolysis or diabetes. Conclusions: Age emerged as a highly significant inverse predictor of good functional outcome after ischemic stroke independent of stroke severity, characteristics, and complications with the age-outcome association exhibiting a nonlinear scale and extending to young stroke patients. Neurology® 2012;78:279–285

Lipoprotein-associated phospholipase A2 activity, ferritin levels, metabolic syndrome, and 10-year cardiovascular and non-cardiovascular mortality: results from the Bruneck study

AIMS To identify factors that influence plasma levels and assess the prognostic value of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in a prospective, population-based survey of the epidemiology and pathogenesis of atherosclerosis. METHODS AND RESULTS The Bruneck study is a prospective, population-based survey initiated in 1990. Lp-PLA2 activity and baseline variables for the current analysis were measured in 765 subjects aged 45-84 years in 1995. Incident cardiovascular disease (CVD) (cardiovascular death, myocardial infarction, stroke, and transient ischaemic attack) and rates of non-CVD mortality were assessed between 1995 and 2005. Subjects with incident CVD had higher levels of Lp-PLA2 activity (884 +/- 196 vs. 771 +/- 192 micromol/min/L, P < 0.001). Increased Lp-PLA2 activity was significantly related to incident CVD [age- and sex-adjusted hazard ratio (95%CI) 2.9 (1.6-5.5); third vs. first tertile group; P < 0.001] and with vascular mortality but not with non-CVD mortality. Lp-PLA2 activity was enhanced in subjects with the metabolic syndrome and showed highly significant positive associations with LDL-C, apoB-100, ferritin, and HOMA-IR, and inverse associations with HDL-C and anti-oxidant levels. CONCLUSION Increased Lp-PLA2 activity is associated with metabolic syndrome and incident fatal and non-fatal CVD, but not with non-CVD mortality. Furthermore, Lp-PLA2 activity is strongly influenced by ferritin levels, LDL-C, and apoB-100 supporting its integral role in lipid peroxidation. Clinical utility of Lp-PLA2 activity for prediction of cardiovascular risk has to be explored in future studies.

CD4+CD8+TCRlow thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid‐induced apoptosis

While signaling by either the TCR or glucocorticoid receptor (GR) can induce apoptosis in thymocytes, recent studies have shown that combining these signals results in survival of CD4+CD8+ thymocytes. Although glucocorticoids (GC) in this way may directly affect T cell selection, no data are available addressing GR expression in thymocyte subsets and in individual cellswithin subsets. We studied GR expression by combining immunofluorescence cell surface staining for CD4, CD8 and TCR with intracellular staining of GR in four‐color cytometry. Significant differences of GR expression were observed in various thymocyte subsets, although a homogeneous distribution of GR expression in individual thymocyte subsets emerged. The highest GR expression was found in CD4–CD8–TCR– thymocytes, and decreased during development via the CD4–CD8+TCR– subpopulation into the CD4+CD8+TCRlow subset. Interestingly, the latter population, although expressing less than half the GR density of CD4–CD8–TCR– cells, is the most sensitive subset to GC‐in‐duced apoptosis. Up‐regulation of TCR expression by the CD4+CD8+TCRlow subset to CD4+CD8+TCRhigh cells was accompanied by a parallel increase in GR expression. The latter finding and the presence of a homogeneous distribution of GR in each thymocyte subset provides an experimental basis for the concept that GR can antagonize TCR‐mediated signals at a constant rate relative to TCR expression.

Early clinical worsening in patients with TIA or minor stroke: The Austrian Stroke Unit Registry

Objective: TIA is associated with a substantial short-term risk of stroke and is thus increasingly recognized as an unstable condition necessitating full medical attention. Our study sought to assess the rate of and predictors for early deterioration after TIA or minor stroke in a large nationwide survey among Austrian stroke units. Methods: Of the 29,287 patients prospectively enrolled in the Austrian Stroke Unit Registry (2003–2008), 8,291 presenting with a TIA or minor ischemic stroke, defined by an NIH Stroke Scale (NIHSS) score <4, were included in the current evaluation. Worsening was defined as clinical deterioration during stroke unit stay by ≥2 points on the NIHSS. Results: A total of 374 patients (4.5%) experienced early clinical worsening during a mean stroke unit stay of 2.97 days (median 2 [interquartile range,1–4] days). In a multivariate stepwise regression analysis hypertension, diabetes, cardiac decompensation, acute infection, and stroke etiology emerged as independent risk predictors for early deterioration. The ABCD2 score could be estimated in a subgroup of 3,886 subjects and closely correlated with the risk of neurologic worsening. Conclusions: Our study revealed a high rate of early clinical deterioration (4.5%) among 8,291 patients with TIA or minor stroke despite immediate admission to specialized stroke units. Predictors for neurologic deterioration apart from diabetes, hypertension, and the estimated ABCD2 score were stroke etiology, reinforcing the relevance of an immediate diagnostic workup, and coexistent acute infection and cardiac decompensation, both conditions necessitating adequate attention in the emergency setting.