Carotid plaque age is a feature of plaque stability inversely related to levels of plasma insulin

Background

The stability of atherosclerotic plaques determines the risk for rupture, which may lead to thrombus formation and potentially severe clinical complications such as myocardial infarction and stroke. Although the rate of plaque formation may be important for plaque stability, this process is not well understood. We took advantage of the atmospheric ¹⁴C-declination curve (a result of the atomic bomb tests in the 1950s and 1960s) to determine the average biological age of carotid plaques.

Methodology/Principal Finding

The cores of carotid plaques were dissected from 29 well-characterized, symptomatic patients with carotid stenosis and analyzed for ¹⁴C content by accelerator mass spectrometry. The average plaque age (i.e. formation time) was 9.6±3.3 years. All but two plaques had formed within 5–15 years before surgery. Plaque age was not associated with the chronological ages of the patients but was inversely related to plasma insulin levels (p = 0.0014). Most plaques were echo-lucent rather than echo-rich (2.24±0.97, range 1–5). However, plaques in the lowest tercile of plaque age (most recently formed) were characterized by further instability with a higher content of lipids and macrophages (67.8±12.4 vs. 50.4±6.2, p = 0.00005; 57.6±26.1 vs. 39.8±25.7, p<0.0005, respectively), less collagen (45.3±6.1 vs. 51.1±9.8, p<0.05), and fewer smooth muscle cells (130±31 vs. 141±21, p<0.05) than plaques in the highest tercile. Microarray analysis of plaques in the lowest tercile also showed increased activity of genes involved in immune responses and oxidative phosphorylation.

Conclusions/Significance

Our results show, for the first time, that plaque age, as judge by relative incorporation of ¹⁴C, can improve our understanding of carotid plaque stability and therefore risk for clinical complications. Our results also suggest that levels of plasma insulin might be involved in determining carotid plaque age.     

Pericardial fat and myocardial perfusion in asymptomatic adults from the Multi-Ethnic Study of Atherosclerosis

Background

Pericardial fat has adverse effects on the surrounding vasculature. Previous studies suggest that pericardial fat may contribute to myocardial ischemia in symptomatic individuals. However, it is unknown if pericardial fat has similar effects in asymptomatic individuals.

Methods

We determined the association between pericardial fat and myocardial blood flow (MBF) in 214 adults with no prior history of cardiovascular disease from the Minnesota field center of the Multi-Ethnic Study of Atherosclerosis (43% female, 56% Caucasian, 44% Hispanic). Pericardial fat volume was measured by computed tomography. MBF was measured by MRI at rest and during adenosine-induced hyperemia. Myocardial perfusion reserve (PR) was calculated as the ratio of hyperemic to resting MBF.

Results

Gender-stratified analyses revealed significant differences between men and women including less pericardial fat (71.9±31.3 vs. 105.2±57.5 cm³, p<0.0001) and higher resting MBF (1.12±0.23 vs. 0.93±0.19 ml/min/g, p<0.0001), hyperemic MBF (3.49±0.76 vs. 2.65±0.72 ml/min/g, p<0.0001), and PR (3.19±0.78 vs. 2.93±0.89, p = 0.03) in women. Correlations between pericardial fat and clinical and hemodynamic variables were stronger in women. In women only (p = 0.01 for gender interaction) higher pericardial fat was associated with higher resting MBF (p = 0.008). However, this association was attenuated after accounting for body mass index or rate-pressure product. There were no significant associations between pericardial fat and hyperemic MBF or PR after multivariate adjustment in either gender. In logistic regression analyses there was also no association between impaired coronary vasoreactivity, defined as having a PR <2.5, and pericardial fat in men (OR, 1.18; 95% CI, 0.82–1.70) or women (OR, 1.11; 95% CI, 0.68–1.82).

Conclusions

Our data fail to support an independent association between pericardial fat and myocardial perfusion in adults without symptomatic cardiovascular disease. Nevertheless, these findings highlight potentially important differences between asymptomatic and symptomatic individuals with respect to the underlying subclinical disease burden.     

Endothelial surface layer degradation by chronic hyaluronidase infusion induces proteinuria in apolipoprotein E-deficient mice

Objective

Functional studies show that disruption of endothelial surface layer (ESL) is accompanied by enhanced sensitivity of the vasculature towards atherogenic stimuli. However, relevance of ESL disruption as causal mechanism for vascular dysfunction remains to be demonstrated. We examined if loss of ESL through enzymatic degradation would affect vascular barrier properties in an atherogenic model.

Methods

Eight week old male apolipoprotein E deficient mice on Western-type diet for 10 weeks received continuous active or heat-inactivated hyaluronidase (10 U/hr, i.v.) through an osmotic minipump during 4 weeks. Blood chemistry and anatomic changes in both macrovasculature and kidneys were examined.

Results

Infusion with active hyaluronidase resulted in decreased ESL (0.32±0.22 mL) and plasma volume (1.03±0.18 mL) compared to inactivated hyaluronidase (0.52±0.29 mL and 1.28±0.08 mL, p<0.05 respectively).Active hyaluronidase increased proteinuria compared to inactive hyaluronidase (0.27±0.02 vs. 0.15±0.01 µg/µg protein/creatinin, p<0.05) without changes in glomerular morphology or development of tubulo-interstitial inflammation. Atherosclerotic lesions in the aortic branches showed increased matrix production (collagen, 32±5 vs. 18±3%; glycosaminoglycans, 11±5 vs. 0.1±0.01%, active vs. inactive hyaluronidase, p<0.05).

Conclusion

ESL degradation in apoE deficient mice contributes to reduced increased urinary protein excretion without significant changes in renal morphology. Second, the induction of compositional changes in atherogenic plaques by hyaluronidase point towards increased plaque vulnerability. These findings support further efforts to evaluate whether ESL restoration is a valuable target to prevent (micro) vascular disease progression.     

Association of the sirtuin and mitochondrial uncoupling protein genes with carotid plaque

Objective

Sirtuins (SIRTs) and mitochondrial uncoupling proteins (UCPs) have been implicated in cardiovascular diseases through the control of reactive oxygen species production. This study sought to investigate the association between genetic variants in the SIRT and UCP genes and carotid plaque.

Methods

In a group of 1018 stroke-free subjects from the Northern Manhattan Study with high-definition carotid ultrasonography and genotyping, we investigated the associations of 85 single nucleotide polymorphisms (SNPs) in the 11 SIRT and UCP genes with the presence and number of carotid plaques, and evaluated interactions of SNPs with sex, smoking, diabetes and hypertension as well as interactions between SNPs significantly associated with carotid plaque.

Results

Overall, 60% of subjects had carotid plaques. After adjustment for demographic and vascular risk factors, T-carriers of the SIRT6 SNP rs107251 had an increased risk for carotid plaque (odds ratio, OR = 1.71, 95% CI = 1.23–2.37, Bonferroni-corrected p = 0.03) and for a number of plaques (rate ratio, RR = 1.31, 1.18–1.45, Bonferroni-corrected p = 1.4×10⁻⁵), whereas T-carriers of the UCP5 SNP rs5977238 had an decreased risk for carotid plaque (OR = 0.49, 95% CI = 0.32–0.74, Bonferroni-corrected p = 0.02) and plaque number (RR = 0.64, 95% CI = 0.52–0.78, Bonferroni-corrected p = 4.9×10⁻⁴). Some interactions with a nominal p≤0.01 were found between sex and SNPs in the UCP1 and UCP3 gene; between smoking, diabetes, hypertension and SNPs in UCP5 and SIRT5; and between SNPs in the UCP5 gene and the UCP1, SIRT1, SIRT3, SIRT5, and SIRT6 genes in association with plaque phenotypes.

Conclusion

We observed significant associations between genetic variants in the SIRT6 and UCP5 genes and atherosclerotic plaque. We also found potential effect modifications by sex, smoking and vascular risk factors of the SIRT/UCP genes in the associations with atherosclerotic plaque. Further studies are needed to validate our observations.     

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model

Purpose

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

Methods

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

Results

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

Conclusion

Pre-confluent cells should be used to maximize graft cell viability.     

Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy

Background

Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.

Methodology/Principal Findings

The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm² respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3⁺ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3⁺/mm² p<0.05), but relative numbers, expressed as a percentage of the total number of CD3⁺ T cells ((FOXP3⁺/CD3⁺) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB⁺ cells were rare in NA, but significantly increased numbers of GrB⁺ cells were found in AV lesions (85±24 and 0.2±0.1 GrB⁺/mm², p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3⁺ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB⁺ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).

Conclusions

Similar to NA, AV is characterized by a low frequency of intimal FOXP3⁺ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.     

Abnormal pulmonary artery stiffness in pulmonary arterial hypertension: in vivo study with intravascular ultrasound

Background

There is increasing recognition that pulmonary artery stiffness is an important determinant of right ventricular (RV) afterload in pulmonary arterial hypertension (PAH). We used intravascular ultrasound (IVUS) to evaluate the mechanical properties of the elastic pulmonary arteries (PA) in subjects with PAH, and assessed the effects of PAH-specific therapy on indices of arterial stiffness.

Method

Using IVUS and simultaneous right heart catheterisation, 20 pulmonary segments in 8 PAH subjects and 12 pulmonary segments in 8 controls were studied to determine their compliance, distensibility, elastic modulus and stiffness index β. PAH subjects underwent repeat IVUS examinations after 6-months of bosentan therapy.

Results

At baseline, PAH subjects demonstrated greater stiffness in all measured indices compared to controls: compliance (1.50±0.11×10^–2 mm^2/mmHg vs 4.49±0.43×10^–2 mm^2/mmHg, p<0.0001), distensibility (0.32±0.03%/mmHg vs 1.18±0.13%/mmHg, p<0.0001), elastic modulus (720±64 mmHg vs 198±19 mmHg, p<0.0001), and stiffness index β (15.0±1.4 vs 11.0±0.7, p = 0.046). Strong inverse exponential associations existed between mean pulmonary artery pressure and compliance (r² = 0.82, p<0.0001), and also between mean PAP and distensibility (r² = 0.79, p = 0.002). Bosentan therapy, for 6-months, was not associated with any significant changes in all indices of PA stiffness.

Conclusion

Increased stiffness occurs in the proximal elastic PA in patients with PAH and contributes to the pathogenesis RV failure. Bosentan therapy may not be effective at improving PA stiffness.     

RANTES/CCL5 and risk for coronary events: results from the MONICA/KORA Augsburg case-cohort, Athero-Express and CARDIoGRAM studies

Background

The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events.

Methods and Findings

We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75–1.42] and 1.11 [0.81–1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years).

Conclusions

High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies.     

Arterial structure and function in mild primary hyperparathyroidism is not directly related to parathyroid hormone, calcium, or vitamin D

Objective

Elevated levels of calcium and parathyroid hormone (PTH), characteristics of primary hyperparathyroidism (PHPT), may be associated with cardiovascular morbidity and mortality in the general population. We evaluated the possible vascular effects of these risk factors in patients with mild PHPT by using standard methods and new imaging techniques.

Design

A prospective case-control study.

Subjects and Methods

Forty-eight patients with mild PHPT without any known cardiovascular risk factors were studied at baseline and at one year after parathyroidectomy (PTX) in comparison with 48 healthy age- and gender-matched controls. We measured biochemical variables, augmentation index (AIx), aortic pulse wave velocity (PWV_ao), radial (IMT_rad) and common carotid artery (IMT_cca) intima media thicknesses, and the grayscale median (IM-GSM) of the latter.

Results

No significant differences were observed between PHPT patients and controls at baseline for AIx (28.6±12.2 vs. 27.7±12.8%), IMT_rad (0.271±0.060 vs. 0.255±0.053 mm), IMT_cca (0.688±0.113 vs. 0.680±0.135 mm), or IM-GSM (82.3±17.2 vs. 86.5±15.3), while PWV_ao was slightly higher in patients (8.68±1.50 vs. 8.13±1.55, p<0.05). Systolic blood pressure (SBP), calcium, and PTH were higher in patients compared with controls, and decreased after PTX, while vitamin D was lower in patients and increased after PTX. While AIx, PWV_ao, IMT_rad, and IMT_cca were related to SBP, neither correlated to vitamin D levels. Only PWV_ao correlated weakly to plasma PTH (r = 0.29, p<0.01) and ionized calcium (r = 0.22, p<0.05) but showed no relation when age and SBP were adjusted for.

Conclusion

We found normal arterial function despite high calcium, PTH, and low vitamin D levels, in patients with mild PHPT without cardiovascular risk factors. The cardiovascular risk associated with low vitamin D and/or high PTH and calcium levels may be explained by their coupling to blood pressure and other risk factors rather than direct effects on arterial structure.     

CSF-biomarkers in Olympic boxing: diagnosis and effects of repetitive head trauma

Background

Sports-related head trauma is common but still there is no established laboratory test used in the diagnostics of minimal or mild traumatic brain injuries. Further the effects of recurrent head trauma on brain injury markers are unknown. The purpose of this study was to investigate the relationship between Olympic (amateur) boxing and cerebrospinal fluid (CSF) brain injury biomarkers.

Methods

The study was designed as a prospective cohort study. Thirty Olympic boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in the study. CSF samples were collected by lumbar puncture 1–6 days after a bout and after a rest period for at least 14 days. The controls were tested once. Biomarkers for acute and chronic brain injury were analysed.

Results

NFL (mean ± SD, 532±553 vs 135±51 ng/L p = 0.001), GFAP (496±238 vs 247±147 ng/L p<0.001), T-tau (58±26 vs 49±21 ng/L p<0.025) and S-100B (0.76±0.29 vs 0.60±0.23 ng/L p = 0.03) concentrations were significantly increased after boxing compared to controls. NFL (402±434 ng/L p = 0.004) and GFAP (369±113 ng/L p = 0.001) concentrations remained elevated after the rest period.

Conclusion

Increased CSF levels of T-tau, NFL, GFAP, and S-100B in >80% of the boxers demonstrate that both the acute and the cumulative effect of head trauma in Olympic boxing may induce CSF biomarker changes that suggest minor central nervous injuries. The lack of normalization of NFL and GFAP after the rest period in a subgroup of boxers may indicate ongoing degeneration. The recurrent head trauma in boxing may be associated with increased risk of chronic traumatic brain injury.     

Increased Platelet Reactivity Is Associated with Circulating Platelet-Monocyte Complexes and Macrophages in Human Atherosclerotic Plaques

Objective

Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs) and macrophages in human atherosclerotic carotid plaques.

Methods

Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP), in two independent cohorts: the Circulating Cells cohort (n = 244) and the Athero-Express cohort (n = 91). Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort). Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort).

Results

We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range): 4153 (1585–11267) area under the curve (AUC) vs. 9633 (3580–21565) AUC, P<0.001). Also, we observed increased platelet reactivity in patients with high macrophage levels in atherosclerotic plaques as compared to patients with low macrophage levels in atherosclerotic plaques (mean±SD; 8969±3485 AUC vs. 7020±3442 AUC, P = 0.02). All associations remained significant after adjustment for age, sex and use of drugs against platelet activation.

Conclusion

Platelet reactivity towards ADP is associated with levels of PMCs and macrophages in human atherosclerotic carotid plaques.     

Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide

Background

Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.

Methods and Findings

In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA₂, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).

Conclusions

Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.     

Clinical outcome of high-risk patients with severe aortic stenosis and reduced left ventricular ejection fraction undergoing medical treatment or TAVI

Introduction

Reduced left ventricular function in patients with severe symptomatic valvular aortic stenosis is associated with impaired clinical outcome in patients undergoing surgical aortic valve replacement (SAVR). Transcatheter Aortic Valve Implantation (TAVI) has been shown non-inferior to SAVR in high-risk patients with respect to mortality and may result in faster left ventricular recovery.

Methods

We investigated clinical outcomes of high-risk patients with severe aortic stenosis undergoing medical treatment (n = 71) or TAVI (n = 256) stratified by left ventricular ejection fraction (LVEF) in a prospective single center registry.

Results

Twenty-five patients (35%) among the medical cohort were found to have an LVEF≤30% (mean 26.7±4.1%) and 37 patients (14%) among the TAVI patients (mean 25.2±4.4%). Estimated peri-interventional risk as assessed by logistic EuroSCORE was significantly higher in patients with severely impaired LVEF as compared to patients with LVEF>30% (medical/TAVI 38.5±13.8%/40.6±16.4% versus medical/TAVI 22.5±10.8%/22.1±12.8%, p <0.001). In patients undergoing TAVI, there was no significant difference in the combined endpoint of death, myocardial infarction, major stroke, life-threatening bleeding, major access-site complications, valvular re-intervention, or renal failure at 30 days between the two groups (21.0% versus 27.0%, p = 0.40). After TAVI, patients with LVEF≤30% experienced a rapid improvement in LVEF (from 25±4% to 34±10% at discharge, p = 0.002) associated with improved NYHA functional class at 30 days (decrease ≥1 NYHA class in 95%). During long-term follow-up no difference in survival was observed in patients undergoing TAVI irrespective of baseline LVEF (p = 0.29), whereas there was a significantly higher mortality in medically treated patients with severely reduced LVEF (log rank p = 0.001).

Conclusion

TAVI in patients with severely reduced left ventricular function may be performed safely and is associated with rapid recovery of systolic left ventricular function and heart failure symptoms.     

Warfarin anticoagulation exacerbates the risk of hemorrhagic transformation after rt-PA treatment in experimental stroke: therapeutic potential of PCC

Background

Oral anticoagulant therapy (OAT) with warfarin is the standard of stroke prevention in patients with atrial fibrillation. Approximately 30% of patients with cardioembolic strokes are on OAT at the time of symptom onset. We investigated whether warfarin exacerbates the risk of thrombolysis-associated hemorrhagic transformation (HT) in a mouse model of ischemic stroke.

Methods

62 C57BL/6 mice were used for this study. To achieve effective anticoagulation, warfarin was administered orally. We performed right middle cerebral artery occlusion (MCAO) for 3 h and assessed functional deficit and HT blood volume after 24 h.

Results

In non-anticoagulated mice, treatment with rt-PA (10 mg/kg i.v.) after 3 h MCAO led to a 5-fold higher degree of HT compared to vehicle-treated controls (4.0±0.5 µl vs. 0.8±0.1, p<0.001). Mice on warfarin revealed larger amounts of HT after rt-PA treatment in comparison to non-anticoagulated mice (9.2±3.2 µl vs. 2.8±1.0, p<0.05). The rapid reversal of anticoagulation by means of prothrombin complex concentrates (PCC, 100 IU/kg) at the end of the 3 h MCAO period, but prior to rt-PA administration, neutralized the exacerbated risk of HT as compared to sham-treated controls (3.8±0.7 µl vs. 15.0±3.8, p<0.001).

Conclusion

In view of the vastly increased risk of HT, it seems to be justified to withhold tPA therapy in effectively anticoagulated patients with acute ischemic stroke. The rapid reversal of anticoagulation with PCC prior to tPA application reduces the risk attributed to warfarin pretreatment and may constitute an interesting therapeutic option.     

Habitual physical activity in mitochondrial disease

Purpose

Mitochondrial disease is the most common neuromuscular disease and has a profound impact upon daily life, disease and longevity. Exercise therapy has been shown to improve mitochondrial function in patients with mitochondrial disease. However, no information exists about the level of habitual physical activity of people with mitochondrial disease and its relationship with clinical phenotype.

Methods

Habitual physical activity, genotype and clinical presentations were assessed in 100 patients with mitochondrial disease. Comparisons were made with a control group individually matched by age, gender and BMI.

Results

Patients with mitochondrial disease had significantly lower levels of physical activity in comparison to matched people without mitochondrial disease (steps/day; 6883±3944 vs. 9924±4088, p = 0.001). 78% of the mitochondrial disease cohort did not achieve 10,000 steps per day and 48%were classified as overweight or obese. Mitochondrial disease was associated with less breaks in sedentary activity (Sedentary to Active Transitions, % per day; 13±0.03 vs. 14±0.03, p = 0.001) and an increase in sedentary bout duration (bout lengths / fraction of total sedentary time; 0.206±0.044 vs. 0.187±0.026, p = 0.001). After adjusting for covariates, higher physical activity was moderately associated with lower clinical disease burden (steps / day; r_s = −0.49; 95% CI −0.33, −0.63, P<0.01). There were no systematic differences in physical activity between different genotypes mitochondrial disease.

Conclusions

These results demonstrate for the first time that low levels of physical activity are prominent in mitochondrial disease. Combined with a high prevalence of obesity, physical activity may constitute a significant and potentially modifiable risk factor in mitochondrial disease.     

Concomitant active tuberculosis prolongs survival in non-small cell lung cancer: a study in a tuberculosis-endemic country

Background

Adjuvant tumor cell vaccine with chemotherapy against non-small cell lung cancer (NSCLC) shows limited clinical response. Whether it provokes effective cellular immunity in tumor microenvironment is questionable. Concomitant active tuberculosis in NSCLC (TBLC) resembles locoregional immunotherapy of tumor cell vaccine; thus, maximally enriches effective anti-tumor immunity. This study compares the survival and immunological cell profile in TBLC over NSCLC alone.

Methods

Retrospective review of NSCLC patients within 1-year-period of 2007 and follow-up till 2010.

Results

A total 276 NSCLC patients were included. The median survival of TBLC is longer than those of NSCLC alone (11.6 vs. 8.8 month, p<0.01). Active tuberculosis is an independent predictor of better survival with HR of 0.68 (95% CI, 0.48∼0.97). Squamous cell carcinoma (SCC) (55.8 vs. 31.7%, p<0.01) is a significant risk factor for NSCLC with active TB. The median survival of SCC with active tuberculosis is significantly longer than adenocarcinoma or undetermined NSCLC with TB (14.2 vs. 6.6 and 2.8 months, p<0.05). Active tuberculosis in SCC increases the expression of CD3 (46.4±24.8 vs. 24.0±16.0, p<0.05), CXCR3 (35.1±16.4 vs. 19.2±13.3, p<0.01) and IP-10 (63.5±21.9 vs. 35.5±21.0, p<0.01), while expression of FOXP3 is decreased (3.5±0.5 vs. 13.3±3.7 p<0.05, p<0.05). Survival of SCC with high expression of CD3 (12.1 vs. 3.6 month, p<0.05) and CXCR3 (12.1 vs. 4.4 month, p<0.05) is longer than that with low expression.

Conclusions

Active tuberculosis in NSCLC shows better survival outcome. The effective T lymphocyte infiltration in tumor possibly underlies the mechanism. Locoregional immunotherapy of tumor cell vaccine may deserve further researches.     

Blockade of VEGFR1 and 2 suppresses pathological angiogenesis and vascular leakage in the eye

Objective

VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.

Materials and Methods

Neutralizing antibodies specific for mouse VEGFR1 (MF1) and VEGFR2 (DC101) were administrated systemically. CNV was induced by laser photocoagulation and assessed 14d after laser treatment. Retinal NV was generated in oxygen-induced ischemic retinopathy (OIR) and assessed at p17. NV quantification was determined by measuring NV tufts and vascular leakage was quantified by measuring [³H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.

Results

VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.

Conclusion

Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents.     

Osteoprotegerin,Pericytes and Bone-Like Vascular Calcification Are Associated with Carotid Plaque Stability

Background and Purpose

Vascular calcification, recapitulating bone formation, has a profound impact on plaque stability. The aim of the present study was to determine the influence of bone-like vascular calcification (named osteoid metaplasia = OM) and of osteoprotegerin on plaque stability.

Methods

Tissue from carotid endarterectomies were analysed for the presence of calcification and signs of vulnerability according to AHA grading system. Osteoprotegerin (OPG), pericytes and endothelial cells were sought using immuno-histochemistry. Symptoms and preoperative imaging findings (CT-scan, MRI and Doppler-scan) were analyzed. Human pericytes were cultured to evaluate their ability to secrete OPG and to influence mineralization in the plaque.

Results

Seventy-three carotid plaques (49 asymptomatic and 24 symptomatic) were harvested. A significantly higher presence of OM (18.4% vs 0%, p<0.01), OPG (10.2% of ROI vs 3.4% of ROI, p<0.05) and pericytes (19% of ROI vs 3.8% of ROI, p<0.05) were noted in asymptomatic compared to symptomatic plaques. Consistently, circulating OPG levels were higher in the plasma of asymptomatic patients (3.2 ng/mL vs 2.5 ng/mL, p = 0.05). In vitro, human vascular pericytes secreted considerable amounts of OPG and underwent osteoblastic differentiation. Pericytes also inhibited the osteoclastic differentiation of CD14+ cells through their secretion of OPG.

Conclusions

OPG (intraplaque an plasmatic) and OM are associated with carotid plaque stability. Pericytes may be involved in the secretion of intraplaque OPG and in the formation of OM.     

Brain-derived neurotrophic factor gene Val66Met polymorphism modulates reversible cerebral vasoconstriction syndromes

Background

Reversible cerebral vasoconstriction syndrome (RCVS) could be complicated by cerebral ischemic events. Hypothetical mechanisms of RCVS involve endothelial dysfunction and sympathetic overactivity, both of which were reported to be related to brain-derived neurotrophic factor (BDNF). The study investigated the association between functional BDNF Val66Met polymorphism and RCVS.

Methods

Patients with RCVS and controls were prospectively recruited and genotyped for the BDNF Val66Met polymorphism. Magnetic resonance angiography (MRA) and transcranial color-coded Doppler sonography were employed to evaluate cerebral vasoconstriction. Genotyping results, clinical parameters, vasoconstriction scores, mean flow velocities of the middle cerebral artery (V_MCA), and Lindegaard indices were analyzed. Split-sample approach was employed to internally validate the data.

Principal Findings

Ninety Taiwanese patients with RCVS and 180 age- and gender-matched normal controls of the same ethnicity completed the study. The genotype frequencies did not differ between patients and controls. Compared to patients with Met/Met homozygosity, patients with Val allele had higher mean vasoconstriction scores of all arterial segments (1.60±0.72 vs. 0.87±0.39, p<0.001), V_MCA values (116.7±36.2 vs. 82.7±17.9 cm/s, p<0.001), and LI (2.41±0.91 vs. 1.89±0.41, p = 0.001). None of the Met/Met homozygotes, but 38.9% of the Val carriers, had V_MCA values of >120 cm/s (p<0.001). Split-sample validation by randomization, age, entry time or residence of patients demonstrated concordant findings.

Conclusions

Our findings link BDNF Val66Met polymorphism with the severity of RCVS for the first time and implicate possible pathogenic mechanisms for vasoconstriction in RCVS.