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.     

Maternal arterial stiffness in women who subsequently develop pre-eclampsia

Background/Objectives

Pre-eclampsia (PE) is associated with profound changes in the maternal cardiovascular system. The aim of the present study was to assess whether alterations in the maternal arterial stiffness precede the onset of PE in at risk women.

Methodology/Principal Findings

This was a cross sectional study involving 70 pregnant women with normal and 70 women with abnormal uterine artery Doppler examination at 22–24 weeks of gestation. All women had their arterial stiffness (augmentation index and pulse wave velocity of the carotid-femoral and carotid-radial parts of the arterial tree) assessed by applanation tonometry in the second trimester of pregnancy, at the time of the uterine artery Doppler imaging. Among the 140 women participating in the study 29 developed PE (PE group) and 111 did not (non-PE group). Compared to the non-PE group, women that developed PE had higher central systolic (94.9±8.6 mmHg vs 104.3±11.1 mmHg; p = <0.01) and diastolic (64.0±6.0 vs 72.4±9.1; p<0.01) blood pressures. All the arterial stiffness indices were adjusted for possible confounders and expressed as multiples of the median (MoM) of the non-PE group. The adjusted median augmentation index was similar between the two groups (p = 0.84). The adjusted median pulse wave velocities were higher in the PE group compared to the non-PE group (carotid-femoral: 1.10±0.14 MoMs vs 0.99±0.11 MoMs; p<0.01 and carotid-radial: 1.08±0.12 MoMs vs 1.0±0.11 MoMs; p<0.01).

Conclusions/Significance

Increased maternal arterial stiffness, as assessed by pulse wave velocity, predates the development of PE in at risk women.     

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.     

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.     

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.     

Argon Inhalation Attenuates Retinal Apoptosis after Ischemia/Reperfusion Injury in a Time- and Dose-Dependent Manner in Rats

Purpose

Retinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat''s eye.

Methods

IRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA.

Results

IRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%.

Conclusion

Immediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon''s possible role as a therapeutic option.     

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.     

Exercise-induced splanchnic hypoperfusion results in gut dysfunction in healthy men

Background

Splanchnic hypoperfusion is common in various pathophysiological conditions and often considered to lead to gut dysfunction. While it is known that physiological situations such as physical exercise also result in splanchnic hypoperfusion, the consequences of flow redistribution at the expense of abdominal organs remained to be determined. This study focuses on the effects of splanchnic hypoperfusion on the gut, and the relationship between hypoperfusion, intestinal injury and permeability during physical exercise in healthy men.

Methods and Findings

Healthy men cycled for 60 minutes at 70% of maximum workload capacity. Splanchnic hypoperfusion was assessed using gastric tonometry. Blood, sampled every 10 minutes, was analyzed for enterocyte damage parameters (intestinal fatty acid binding protein (I-FABP) and ileal bile acid binding protein (I-BABP)). Changes in intestinal permeability were assessed using sugar probes. Furthermore, liver and renal parameters were assessed. Splanchnic perfusion rapidly decreased during exercise, reflected by increased gap_g-apCO₂ from −0.85±0.15 to 0.85±0.42 kPa (p<0.001). Hypoperfusion increased plasma I-FABP (615±118 vs. 309±46 pg/ml, p<0.001) and I-BABP (14.30±2.20 vs. 5.06±1.27 ng/ml, p<0.001), and hypoperfusion correlated significantly with this small intestinal damage (r_S = 0.59; p<0.001). Last of all, plasma analysis revealed an increase in small intestinal permeability after exercise (p<0.001), which correlated with intestinal injury (r_S = 0.50; p<0.001). Liver parameters, but not renal parameters were elevated.

Conclusions

Exercise-induced splanchnic hypoperfusion results in quantifiable small intestinal injury. Importantly, the extent of intestinal injury correlates with transiently increased small intestinal permeability, indicating gut barrier dysfunction in healthy individuals. These physiological observations increase our knowledge of splanchnic hypoperfusion sequelae, and may help to understand and prevent these phenomena in patients.     

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.     

Humanin, a Cytoprotective Peptide, Is Expressed in Carotid Artherosclerotic Plaques in Humans

Objective

The mechanism of atherosclerotic plaque progression leading to instability, rupture, and ischemic manifestation involves oxidative stress and apoptosis. Humanin (HN) is a newly emerging endogenously expressed cytoprotective peptide. Our goal was to determine the presence and localization of HN in carotid atherosclerotic plaques.

Methods and Results

Plaque specimens from 34 patients undergoing carotid endarterectomy were classified according to symptomatic history. Immunostaining combined with digital microscopy revealed greater expression of HN in the unstable plaques of symptomatic compared to asymptomatic patients (29.42±2.05 vs. 14.14±2.13% of plaque area, p<0.0001). These data were further confirmed by immunoblot (density of HN/β-actin standard symptomatic vs. asymptomatic 1.32±0.14 vs. 0.79±0.11, p<0.01). TUNEL staining revealed a higher proportion of apoptotic nuclei in the plaques of symptomatic patients compared to asymptomatic (68.25±3.61 vs. 33.46±4.46% of nuclei, p<0.01). Double immunofluorescence labeling revealed co-localization of HN with macrophages (both M1 and M2 polarization), smooth muscle cells, fibroblasts, and dendritic cells as well as with inflammatory markers MMP2 and MMP9.

Conclusions

The study demonstrates a higher expression of HN in unstable carotid plaques that is localized to multiple cell types within the plaque. These data support the involvement of HN in atherosclerosis, possibly as an endogenous response to the inflammatory and apoptotic processes within the atheromatous plaque.     

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.     

Augmented atherogenesis in LDL receptor deficient mice lacking both macrophage ABCA1 and ApoE

Aim

ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO''s, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10³ µm²), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10³ µm²; apoE KO: 402±78×10³ µm², respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10³ µm²). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).

Conclusions

Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.     

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.     

Activated Protein C Has a Protective Effect against Myocardial I/R Injury by Improvement of Endothelial Function and Activation of AKT1

Objectives

Activated protein C (APC) has a protective efficacy against ischemia-reperfusion (I/R) injury in several organs. The objective of this study was to investigate effect of APC in myocardium with possible mechanism.

Methods

We used regional and global myocardial I/R injury models of rats. They consisted of I/R injuries (1) by ligation of left coronary artery, or (2) using Langendorff apparatus. Langendorff was used to focus the mechanism of APC excluding coagulation cascade in a working heart. Each experiment had an APC group (n = 10) and a control group with normal saline (n = 10). Injections of these solutions into rats were performed 30 minutes before the planned-I/R injury. Cardiac performance after the procedure was evaluated by echocardiography or indices with Langendorff apparatus. Coronary flow (CF) was measured in the global I/R injury model. Western blotting was performed to detect the change of AKT1 signal in myocardium after global I/R injury.

Results

LV function improved significantly in the APC group: %EF at 2 weeks after procedure, 70.8%±4.5% vs. 56.5%±0.7%; APC vs. control; p<0.01. Percent LV development pressure (LVDP) also improved in the APC group significantly, 88.8%±45.3% vs. 28.1%±15.4%; APC vs. control; p<0.01. In APC group, %CF improved significantly, 88.5%±15.8% vs. 65.0%±13.4%; APC vs. control; p<0.01. It was enhanced significantly when acetylcholine was administered; % CF: 103.5%±9.9% vs. 87.0%±12.1%; APC vs. control; p<0.05. Western blotting revealed that APC significantly induced activation of phosphorylated AKT1 in myocardium (p<0.05).

Conclusions

APC has a novel effect to protect myocardium and cardiac performance against I/R injury through improvement of endothelial function and activation of AKT1.     

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.     

The effect of prolonged physical activity performed during extreme caloric deprivation on cardiac function

Background

Endurance exercise may induce transient cardiac dysfunction. Data regarding the effect of caloric restriction on cardiac function is limited. We studied the effect of physical activity performed during extreme caloric deprivation on cardiac function.

Methods

Thirty-nine healthy male soldiers (mean age 20±0.3 years) were studied during a field training exercise lasted 85–103 hours, with negligible food intake and unlimited water supply. Anthropometric measurements, echocardiographic examinations and blood and urine tests were performed before and after the training exercise.

Results

Baseline VO₂ max was 59±5.5 ml/kg/min. Participants'' mean weight reduction was 5.7±0.9 kg. There was an increase in plasma urea (11.6±2.6 to 15.8±3.8 mmol/L, p<0.001) and urine osmolarity (692±212 to 1094±140 mmol/kg, p<0.001) and a decrease in sodium levels (140.5±1.0 to 136.6±2.1 mmol/L, p<0.001) at the end of the study. Significant alterations in diastolic parameters included a decrease in mitral E wave (93.6 to 83.5 cm/s; p = 0.003), without change in E/A and E/E′ ratios, and an increase in iso-volumic relaxation time (73.9 to 82.9 ms, p = 0.006). There was no change in left or right ventricular systolic function, or pulmonary arterial pressure. Brain natriuretic peptide (BNP) levels were significantly reduced post-training (median 9 to 0 pg/ml, p<0.001). There was no elevation in Troponin T or CRP levels. On multivariate analysis, BNP reduction correlated with sodium levels and weight reduction (R = 0.8, p<0.001).

Conclusions

Exposure to prolonged physical activity performed under caloric deprivation resulted in minor alterations of left ventricular diastolic function. BNP levels were significantly reduced due to negative water and sodium balance.     

IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage

Background

Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied.

Methodology/Principal Findings

Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s⁻¹ over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA.

Conclusions/Significant Findings

We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.     

Effects of Stimulation of Soluble Guanylate Cyclase on Diabetic Nephropathy in Diabetic eNOS Knockout Mice on Top of Angiotensin II Receptor Blockade

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy.Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks.Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress.In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.     

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.