Obstructive Sleep Apnoea Syndrome,Endothelial Function and Markers of Endothelialization. Changes after CPAP

Study objectives

This study tries to assess the endothelial function in vivo using flow-mediated dilatation (FMD) and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA) syndrome at baseline and after three months with CPAP therapy.

Design

Observational study, before and after CPAP therapy.

Setting and Patients

We studied 30 patients with apnoea/hypopnoea index (AHI) >15/h that were compared with themselves after three months of CPAP therapy. FMD was assessed non-invasively in vivo using the Laser-Doppler flowmetry. Circulating cell-free DNA (cf-DNA) and microparticles (MPs) were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF) was determined as a marker of endothelial restoration process.

Measurements and results

After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, p< 0.005) cf-DNA and MPs significantly decreased (187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p<0.01, and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/μL, p<0.05, respectively) and VEGF levels increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p<0.05). These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p<0.005) but not between markers of damage and restoration, thus suggesting that both types of markers should be measured together.

Conclusions

CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage.     

Skin Autofluorescence Is Associated with Endothelial Dysfunction in Uremic Subjects on Hemodialysis

Background

Elevated levels of advanced glycation end products (AGEs) within tissues may contribute to endothelial dysfunction, an early indicator of atherosclerosis. We aimed to investigate whether levels of skin AGEs could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.

Methods and Results

One hundred and nineteen uremic patients on hemodialysis and 57 control subjects with moderate-to-high cardiovascular risk factors and without chronic kidney disease (CKD) were enrolled. We used ultrasound to measure flow-mediated vasodilation (FMD). An AGE reader measured skin autoflurorescence (AF). We then compared differences in FMD and skin AF values between the two groups. The uremic subjects had significantly higher levels of skin AF (3.47±0.76 AU vs. 2.21±0.45 arbitrary units; P<0.01) and significantly lower levels of FMD (4.79%±1.88% vs. 7.19%±2.17%; P<0.01) than the non-CKD subjects. After adjusting for all potential covariates, we found that skin AF level independently predicted FMD in both the hemodialysis and the non-CKD groups. In the hemodialysis group, skin AF ≥ 3.05 arbitrary units predicted abnormal FMD at a sensitivity of 87.9% and a specificity of 78.6% (P<0.01).

Conclusions

Skin AF could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.     

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

Purpose

Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.

Methods

To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.

Results

Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.

Conclusion

Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.     

Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

Objective

Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).

Methods

Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.

Results

ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.

Conclusions

Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.     

Inflammation,Endothelial Dysfunction and Increased Left Ventricular Mass in Chronic Kidney Disease (CKD) Patients: A Longitudinal Study

Introduction

Within this longitudinal study we investigated the association of inflammation markers C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) and endothelial dysfunction markers intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) with left ventricular mass indexed for height²·⁷¹ (LVMI) in hypertensive predialysis CKD patients.

Material and Methods

From 2004 to 2005, 182 incident consecutive adult patients from the outpatient CKD clinics of two hospitals in Greece with CKD and hypertension or using antihypertensive medication, were included. Of these, 107 patients underwent CRP (mg/l) and LVMI (g/height²·⁷¹) measurements annually for three years.

Results

In the longitudinal analyses, using linear mixed modeling, a higher IL-6 (ß = 1.9 (95%ci:0.38;3.5), inflammation score based on CRP, IL-6 and TNF-α (ß = 5.0 (95%ci:0.72; 9.4) and VCAM-1 (ß = 0.01 (95%ci:0.005;0.02) were associated with higher LVMI. These models were adjusted for age, gender and primary renal disease, and for confounders that on top changed the beta with ≥10%, i.e. diuretic use (for IL-6 and inflammation score).

Conclusion

The results suggest that in predialysis CKD patients, inflammation as well as endothelial dysfunction may play an important role towards the increase in LVMI.     

Switching Patients with Non-Dialysis Chronic Kidney Disease from Oral Iron to Intravenous Ferric Carboxymaltose: Effects on Erythropoiesis-Stimulating Agent Requirements,Costs, Hemoglobin and Iron Status

Background

Patients with non-dialysis-dependent chronic kidney disease (ND-CKD) often receive an erythropoiesis-stimulating agent (ESA) and oral iron treatment. This study evaluated whether a switch from oral iron to intravenous ferric carboxymaltose can reduce ESA requirements and improve iron status and hemoglobin in patients with ND-CKD.

Methods

This prospective, single arm and single-center study included adult patients with ND-CKD (creatinine clearance ≤40 mL/min), hemoglobin 11–12 g/dL and iron deficiency (ferritin <100 μg/L or transferrin saturation <20%), who were regularly treated with oral iron and ESA during 6 months prior to inclusion. Study patients received an intravenous ferric carboxymaltose dose of 1,000 mg iron, followed by a 6-months ESA/ ferric carboxymaltose maintenance regimen (target: hemoglobin 12 g/dL, transferrin saturation >20%). Outcome measures were ESA dose requirements during the observation period after initial ferric carboxymaltose treatment (primary endpoint); number of hospitalizations and transfusions, renal function before and after ferric carboxymaltose administration, number of adverse reactions (secondary endpoints). Hemoglobin, mean corpuscular volume, ferritin and transferrin saturation were measured monthly from baseline until end of study. Creatinine clearance, proteinuria, C-reactive protein, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase bimonthly from baseline until end of study.

Results

Thirty patients were enrolled (age 70.1±11.4 years; mean±SD). Mean ESA consumption was significantly reduced by 83.2±10.9% (from 41,839±3,668 IU/patient to 6,879±4,271 IU/patient; p<0.01). Hemoglobin increased by 0.7±0.3 g/dL, ferritin by 196.0±38.7 μg/L and transferrin saturation by 5.3±2.9% (month 6 vs. baseline; all p<0.01). No ferric carboxymaltose-related adverse events were reported and no patient withdrew or required transfusions during the study.

Conclusion

Among patients with ND-CKD and stable normal or borderline hemoglobin, switching from oral iron to intravenous ferric carboxymaltose was associated with significant improvements in hematological and iron parameters and a significant reduction in ESA dose requirements in this single-center pilot study.

Trial Registration

ClinicalTrials.gov NCT02232906     

Age- and Gender Dependent Liver Fat Content in a Healthy Normal BMI Population as Quantified by Fat-Water Separating DIXON MR Imaging

Objectives

To establish age- and sex-dependent values of magnetic resonance (MR) liver fat-signal fraction (FSF) in healthy volunteers with normal body-mass index (BMI).

Methods

2-point mDIXON sequences (repetition time/echo time, 4.2msec/1.2msec, 3.1msec) at 3.0 Tesla MR were acquired in 80 healthy volunteers with normal BMI (18.2 to 25.7 kg/m2) between 20 and 62 years (10 men/10 women per decade). FSF was measured in 5 liver segments (segment II, III, VI, VII, VIII) based on mean signal intensities in regions of interest placed on mDIXON-based water and fat images. Multivariate general linear models were used to test for significant differences between BMI-corrected FSF among age subgroups. Pearson and Spearman correlations between FSF and several body measures were calculated.

Results

Mean FSF (%) ± standard deviations significantly differed between women (3.91 ± 1.10) and men (4.69 ± 1.38) and varied with age for women/men (p-value: 0.002/0.027): 3.05 ± 0.49/3.74 ± 0.60 (age group 20–29), 3.75 ± 0.66/4.99 ± 1.30 (30–39), 4.76 ± 1.16/5.25 ± 1.97 (40–49) and 4.09 ± 1.26/4.79 ± 0.93 (50–62). FSF differences among age subgroups were significant for women only (p = 0.003).

Conclusions

MR-based liver fat content is higher in men and peaks in the fifth decade for both genders.     

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

Background & Aims

Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.

Methods

Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.

Results

Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×10⁷ hepatocytes, 1.8±0.5×10⁶ Kupffer cells, 4.3±1.9×10⁵ liver sinusoidal endothelial cells, and 3.2±0.5×10⁵ stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146⁺ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.

Conclusions

Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.     

HbA1c Levels Are Associated with Chronic Kidney Disease in a Non-Diabetic Adult Population: A Nationwide Survey (KNHANES 2011–2013)

Background

Many studies have reported an association between glycated hemoglobin A1c (HbA1c) and metabolic syndrome (MetS) in non-diabetes patients. Each component of MetS is in fact related to chronic kidney disease (CKD) incidence and progression. Therefore, HbA1c in non-diabetic mellitus (DM) may be intrinsically associated with the prevalence of CKD. The hypothesis of the present study was that high HbA1c in non-DM patients is associated with CKD.

Patients and Methods

The total number of participants in this study was 24,594. The participants were divided into three groups according to their HbA1c levels: a Low group (<5.7% or <39 mmol/mol), a Middle group (5.7–6.0% or 39–42 mmol/mol), and a High group (>6.0% or >42 mmol/mol). The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation.

Results

The number of participants allocated to the Low, Middle, and High groups was 8,651, 4,634, and 1,387, respectively. Linear regression analyses were performed to evaluate the association between variables. Standardized β ± standard error was 0.25 ± 0.22 for waist circumference, 0.44 ± 0.20 for fasting glucose, –0.14 ± 0.30 for high-density lipoprotein cholesterol levels, 0.15 ± 2.31 for triglyceride levels, 0.21 ± 0.00 for systolic blood pressure, 0.10 ± 0.00 for diastolic blood pressure, and –0.22 ± 0.42 for eGFR (P < 0.001 for all variables). eGFR in non-diabetes participants was inversely associated with the HbA1c level, where eGFR decreased as HbA1c levels increased. Standardized βs were –0.04 ± 0.42 in multivariable analysis (P < 0.001). The proportion of participants with only MetS, only CKD, or both MetS and CKD was higher in the High group than in the Low and Middle groups.

Conclusion

High HbA1c in non-DM patients may be associated with CKD. Renal function in patients with high HbA1c levels may need to be monitored.     

Hyperglycaemia Enhances Nitric Oxide Production in Diabetes: A Study from South Indian Patients

Background

We have previously reported that increased glucose levels were associated with higher serum nitric oxide (NO) levels in fructose-fed insulin resistant rats. However, the relationship between hyperglycemia and serum NO level was not clear. Therefore, the present study was designed to find the association between hyperglycemia and serum NO levels in Type 2 diabetic (T2DM) patients and T2DM with cardiovascular complication.

Methods

Endothelial cells (HUVEC) were treated with of D-glucose (10-100mM), and NO levels and NOS gene expression was measured. Hyperglycaemia was induced in Sprague-Dawley rats, and serum NO levels were measured after 8 weeks. For clinical evaluation, five groups of patients were recruited: Control (CT, n=48), Type 2 diabetes (T2DM, n=26), T2DM with hypertension (DMHT, n=46), Coronary artery diseases (CAD, n=29) and T2DM with coronary artery diseases (DMCD, n=38). NO (nitrite + nitrate) levels were measured from human serum.

Results

We found a significant (p<0.05) and dose-dependent increase in NO levels in HUVEC cells after 4 hours of high glucose exposure. eNOS and iNOS gene expression was increased in HUVEC cells after different concentrations and time periods of glucose treatment. We also observed significant (149.1±25μM, p<0.01) increase in serum NO levels in hyperglycaemic rats compared to control (76.6±13.2μM). Serum NO level was significantly higher in T2DM (111.8 μM (81.7-122.4), p<0.001) and DMCD patients ((129.4 μM (121.2-143.5), p <0.001) but not in CAD patients (76.4 μM (70.5-87)), as compared to control (68.2 μM (56.4-82.3)). We found significantly lower NO levels (83.5 μM (60.5-122.9)) in subjects suffering from diabetes since more than 5 years, compared to subjects (115.3 μM (75.2-127.1), p<0.001) with less than 5 years.

Conclusion

In conclusion, high NO levels were observed in South Indian diabetic patients. Higher glucose levels in serum might be responsible for activation of endothelial cells to enhance NO levels.     

3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells

Aims

The 3D geometry of individual vascular smooth muscle cells (VSMCs), which are essential for understanding the mechanical function of blood vessels, are currently not available. This paper introduces a new 3D segmentation algorithm to determine VSMC morphology and orientation.

Methods and Results

A total of 112 VSMCs from six porcine coronary arteries were used in the analysis. A 3D semi-automatic segmentation method was developed to reconstruct individual VSMCs from cell clumps as well as to extract the 3D geometry of VSMCs. A new edge blocking model was introduced to recognize cell boundary while an edge growing was developed for optimal interpolation and edge verification. The proposed methods were designed based on Region of Interest (ROI) selected by user and interactive responses of limited key edges. Enhanced cell boundary features were used to construct the cell’s initial boundary for further edge growing. A unified framework of morphological parameters (dimensions and orientations) was proposed for the 3D volume data. Virtual phantom was designed to validate the tilt angle measurements, while other parameters extracted from 3D segmentations were compared with manual measurements to assess the accuracy of the algorithm. The length, width and thickness of VSMCs were 62.9±14.9μm, 4.6±0.6μm and 6.2±1.8μm (mean±SD). In longitudinal-circumferential plane of blood vessel, VSMCs align off the circumferential direction with two mean angles of -19.4±9.3° and 10.9±4.7°, while an out-of-plane angle (i.e., radial tilt angle) was found to be 8±7.6° with median as 5.7°.

Conclusions

A 3D segmentation algorithm was developed to reconstruct individual VSMCs of blood vessel walls based on optical image stacks. The results were validated by a virtual phantom and manual measurement. The obtained 3D geometries can be utilized in mathematical models and leads a better understanding of vascular mechanical properties and function.     

A Comparison of the Effects of the GLP-1 Analogue Liraglutide and Insulin Glargine on Endothelial Function and Metabolic Parameters: A Randomized,Controlled Trial Sapporo Athero-Incretin Study 2 (SAIS2)

Objectives

GLP-1 improves hyperglycemia, and it has been reported to have favorable effects on atherosclerosis. However, it has not been fully elucidated whether GLP-1 is able to improve endothelial function in patients with type 2 diabetes. Therefore, we investigated the efficacy of the GLP-1 analogue, liraglutide on endothelial function and glycemic metabolism compared with insulin glargine therapy.

Materials and Methods

In this multicenter, prospective randomized parallel-group comparison study, 31 diabetic outpatients (aged 60.3 ± 10.3 years with HbA1c levels of 8.6 ± 0.8%) with current metformin and/or sulfonylurea treatment were enrolled and randomly assigned to receive liraglutide or glargine therapy once daily for 14 weeks. Flow mediated dilation (FMD), a comprehensive panel of hemodynamic parameters (Task Force Monitor), and serum metabolic markers were assessed before and after the treatment period.

Results

A greater reduction (worsening) in %FMD was observed in the glargine group, although this change was not statistically different from the liraglutide group (liraglutide; 5.7 to 5.4%, glargine 6.7 to 5.7%). The augmentation index, C-peptide index, derivatives of reactive oxygen metabolites and BMI were significantly improved in the liraglutide group. Central systolic blood pressure and NT-proBNP also tended to be improved in the liraglutide-treated group, while improvements in HbA1c levels were similar between groups. Cardiac index, blood pressure and most other metabolic parameters were not different.

Conclusions

Regardless of glycemic improvement, early liraglutide therapy did not affect endothelial function but may provide favorable effects on beta-cell function and cardioprotection in type 2 diabetics without advanced atherosclerosis.

Trial Registration

UMIN Clinical Trials Registry System as trial ID UMIN000005331.     

Rosuvastatin improves impaired endothelial function,lowers high sensitivity CRP,complement and immuncomplex production in patients with systemic sclerosis - a prospective case-series study

Introduction

We studied the effect of rosuvastatin on endothelial and macrovascular function, cardiovascular risk factors and the complement pathway in patients with systemic sclerosis (SSc).

Methods

Altogether 28 patients with SSc underwent laboratory and complex vascular assessments before and after six months of 20 mg rosuvastatin treatment. Flow-mediated dilation (FMD) of the brachial artery, as well as carotid artery intima-media thickness (ccIMT), carotid-femoral and aorto-femoral pulse wave-velocity (PWV) were analyzed by ECG-synchronized ultrasound. Ankle-brachial index (ABI) was determined by Doppler, and forearm skin microcirculation was assessed by Laser Doppler perfusion monitoring.

Results

Brachial artery FMD significantly improved upon rosuvastatin therapy (2.2% ± 3.3% before versus 5.7% ± 3.9% after treatment, P = 0.0002). With regard to patient subsets, FMD significantly improved in the 21 lcSSc patients (from 2.1% to 5.6%, P = 0.001). In the seven dcSSc patients, we observed a tendency of improvement in FMD (from 3% to 6%, P = 0.25). Changes in PWV, ccIMT and ABI were not significant. Mean triglyceride (1.7 ± 0.97 versus 1.3 ± 0.46 mmol/l, P = 0.0004), total cholesterol (5.3 ± 1.6 mmol/l versus 4.2 ± 1.3 mmol/l, P = 0.0003), low density lipoprotein cholesterol (3.0 ± 1.3 versus 2.2 ± 1.0 mmol/l, P = 0.005) and C-reactive protein levels (CRP) (5.1 ± 5.2 versus 3.4 ± 2.7, P = 0.01) levels significantly decreased after rosuvastatin treatment. Mean C3, C4 and IC levels also decreased significantly as compared to pretreatment values.

Conclusions

Six-month rosuvastatin therapy improves endothelial function and lowers CRP, C3, C4 and IC levels indicating possible favourable effects of this statin on the cardiovascular and immune system in SSc.     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

The Effect of Exercise Intensity on Endothelial Function in Physically Inactive Lean and Obese Adults

Purpose

To examine the effects of exercise intensity on acute changes in endothelial function in lean and obese adults.

Methods

Sixteen lean (BMI <25, age 23±3 yr) and 10 obese (BMI >30, age 26±6 yr) physically inactive adults were studied during 3 randomized admissions [control (C, no exercise), moderate-intensity exercise (M, @ lactate threshold (LT)) and high-intensity exercise (H, midway between LT and VO₂peak) (30 min)]. Endothelial function was assessed by flow-mediated dilation (FMD) at baseline and 1, 2, and 4 h post-exercise.

Results

RM ANCOVA revealed significant main effects for group, time, and group x condition interaction (p<0.05). A diurnal increase in FMD was observed in lean but not obese subjects. Lean subjects exhibited greater increases in FMD than obese subjects (p = 0.0005). In the obese group a trend was observed for increases in FMD at 2- and 4-hr after M (p = 0.08). For lean subjects, FMD was significantly elevated at all time points after H. The increase in FMD after H in lean subjects (3.2±0.5%) was greater than after both C (1.7±0.4%, p = 0.015) and M (1.4±0.4%, p = 0.002). FMD responses of lean and obese subjects significantly differed after C and H, but not after M.

Conclusion

In lean young adults, high-intensity exercise acutely enhances endothelial function, while moderate-intensity exercise has no significant effect above that seen in the absence of exercise. The FMD response of obese adults is blunted compared to lean adults. Diurnal variation should be considered when examining the effects of acute exercise on FMD.     

Interleukin-32β Propagates Vascular Inflammation and Exacerbates Sepsis in a Mouse Model

Background

Inflammation is associated with most diseases, which makes understanding the mechanisms of inflammation vitally important.

Methodology/Principal Findings

Here, we demonstrate a critical function of interleukin-32β (IL-32β) in vascular inflammation. IL-32β is present in tissues from humans, but is absent in rodents. We found that the gene is highly expressed in endothelial cells. Three isoforms of IL-32, named IL-32α, β, and ε, were cloned from human endothelial cells, with IL-32β being the major isoform. Pro-inflammatory cytokines (TNFα and IL-1β) induced IL-32β expression through NF-κB. Conversely, IL-32β propagated vascular inflammation via induction of vascular cell adhesion molecules and inflammatory cytokines. Accordingly, IL-32β increased adhesion of inflammatory cells to activated endothelial cells, a paramount process in inflammation. These results illustrate a positive feedback regulation that intensifies and prolongs inflammation. Importantly, endothelial/hematopoietic expression of IL-32β in transgenic mice elevated inflammation and worsened sepsis. This was demonstrated by significant elevation of leukocyte infiltration and serum levels of TNFα and IL-1β, increased vascular permeability and lung damage, and accelerated animal death. Together, our results reveal an important function of IL-32 in vascular inflammation and sepsis development.

Conclusions/Significance

Our results reveal an important function of IL-32 in vascular inflammation and sepsis development.     

TGF-β1-Mediated Differentiation of Fibroblasts Is Associated with Increased Mitochondrial Content and Cellular Respiration

Objectivs

Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.

Methods

Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.

Results

Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/10⁶ cells vs. 307 ± 9 μg DNA/10⁶ cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O₂/min/10⁶ cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O₂/min/10⁶ cells).

Conclusions

TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.     

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically

Background

Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to ‘classical’ biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied.

Methods

In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces.

Results

Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011).

Conclusion

In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion.     

Absolute Quantification of Human Liver Phosphorus-Containing Metabolites In Vivo Using an Inhomogeneous Spoiling Magnetic Field Gradient

Purpose

Absolute concentrations of high-energy phosphorus (³¹P) metabolites in liver provide more important insight into physiologic status of liver disease compared to resonance integral ratios. A simple method for measuring absolute concentrations of ³¹P metabolites in human liver is described. The approach uses surface spoiling inhomogeneous magnetic field gradient to select signal from liver tissue. The technique avoids issues caused by respiratory motion, chemical shift dispersion associated with linear magnetic field gradients, and increased tissue heat deposition due to radiofrequency absorption, especially at high field strength.

Methods

A method to localize signal from liver was demonstrated using superficial and highly non-uniform magnetic field gradients, which eliminate signal(s) from surface tissue(s) located between the liver and RF coil. A double standard method was implemented to determine absolute ³¹P metabolite concentrations in vivo. 8 healthy individuals were examined in a 3 T MR scanner.

Results

Concentrations of metabolites measured in eight healthy individuals are: γ-adenosine triphosphate (ATP) = 2.44 ± 0.21 (mean ± sd) mmol/l of wet tissue volume, α-ATP = 3.2 ± 0.63 mmol/l, β-ATP = 2.98 ± 0.45 mmol/l, inorganic phosphates (Pi) = 1.87 ± 0.25 mmol/l, phosphodiesters (PDE) = 10.62 ± 2.20 mmol/l and phosphomonoesters (PME) = 2.12 ± 0.51 mmol/l. All are in good agreement with literature values.

Conclusions

The technique offers robust and fast means to localize signal from liver tissue, allows absolute metabolite concentration determination, and avoids problems associated with constant field gradient (linear field variation) localization methods.     

CT Perfusion with Acetazolamide Challenge in C6 Gliomas and Angiogenesis

Background

This study was performed to investigate the correlation between CT perfusion with acetazolamide challenge and angiogenesis in C6 gliomas.

Methods

Thirty-two male Sprague-Dawley rats were evaluated. The rats were divided randomly to four groups: eight rats with orthotopically implanted C6 gliomas at 10-days old (Group A), eight rats with gliomas at 14-days old (Group B), eight rats with gliomas at 18-days old (Group C), eight rats with orthotopically injected normal saline served as controls. CT perfusion was performed before and after administration of acetazolamide. Changes in perfusion parameters due to acetazolamide administration were calculated and analyzed.

Results

Elevated carbon dioxide partial pressure and decreased pH were found in all 32 rats post acetazolamide challenge (P<0.01). Cerebral blood flow_{pre-challenge} was increased in group C (95.0±2.5 ml/100g/min), as compared to group B (80.1±11.3 ml/100g/min) and group A (63.1±2.1 ml/100g/min). Cerebral blood flow percentage changes were detected with a reduction in group C (54.2±4.8%) as compared to controls (111.3±22.2%). Cerebral blood volume _{pre-challenge} was increased in group C (50.8±1.7ml/100g), as compared to group B (45.7±1.9 ml/100g) and group A (38.2±0.8 ml/100g). Cerebral blood volume percentage changes were decreased in group C (23.5±4.6%) as compared to controls (113.5±30.4%). Angiogenesis ratio = [(CD105-MVD) / (FVIII-MVD)] ×100%. Positive correlations were observed between CD105-microvessel density, angiogenesis ratio, vascular endothelial growth factor, proliferation marker and cerebral blood flow_{pre-challenge}, cerebral blood volume _{pre-challenge}. Negative correlations were observed between CD105-microvessel density and cerebral blood flow percentage changes (P<0.01, correlation coefficient r=-0.788), cerebral blood volume percentage changes (P<0.01, r=-0.703). Negative correlations were observed between angiogenesis ratio, vascular endothelial growth factor, proliferation marker and cerebral blood flow percentage changes, cerebral blood volume percentage changes.

Conclusion

Our findings suggest that CT perfusion with challenge can provide new insight into non-invasive assessment of rat C6 glioma angiogenesis.