Addition of endothelial progenitor cells to renal revascularization restores medullary tubular oxygen consumption in swine renal artery stenosis

Renal artery stenosis (RAS) promotes microvascular rarefaction and fibrogenesis, which may eventuate in irreversible kidney injury. We have shown that percutaneous transluminal renal angioplasty (PTRA) or endothelial progenitor cells (EPC) improve renal cortical hemodynamics and function in the poststenotic kidney. The renal medulla is particularly sensitive to hypoxia, yet little is known about reversibility of medullary injury on restoration of renal blood flow. This study was designed to test the hypothesis that PTRA, with or without adjunct EPC delivery to the stenotic kidney, may improve medullary remodeling and tubular function. RAS was induced in 21 pigs using implantation of irritant coils, while another group served as normal controls (n = 7 each). Two RAS groups were then treated 6 wk later with PTRA or both PTRA and EPC. Four weeks later, medullary hemodynamics, microvascular architecture, and oxygen-dependent tubular function of the stenotic kidneys were examined using multidetector computed tomography, microcomputed tomography, and blood oxygenation level-dependent MRI, respectively. Medullary protein expression of vascular endothelial growth factor, endothelial nitric oxide synthase, hypoxia-inducible factor-1α, and NAD(P)H oxidase p47 were determined. All RAS groups showed decreased medullary vascular density and blood flow. However, in RAS+PTRA+EPC animals, EPC were engrafted in tubular structures, oxygen-dependent tubular function was normalized, and fibrosis attenuated, despite elevated expression of hypoxia-inducible factor-1α and sustained downregulation of vascular endothelial growth factor. In conclusion, EPC delivery, in addition to PTRA, restores medullary oxygen-dependent tubular function, despite impaired medullary blood and oxygen supply. These results support further development of cell-based therapy as an adjunct to revascularization of RAS.     

Efferent Pathways in Sodium Overload-Induced Renal Vasodilation in Rats

Hypernatremia stimulates the secretion of oxytocin (OT), but the physiological role of OT remains unclear. The present study sought to determine the involvement of OT and renal nerves in the renal responses to an intravenous infusion of hypertonic saline. Male Wistar rats (280–350 g) were anesthetized with sodium thiopental (40 mg. kg⁻¹, i.v.). A bladder cannula was implanted for collection of urine. Animals were also instrumented for measurement of mean arterial pressure (MAP) and renal blood flow (RBF). Renal vascular conductance (RVC) was calculated as the ratio of RBF by MAP. In anesthetized rats (n = 6), OT infusion (0.03 µg • kg⁻¹, i.v.) induced renal vasodilation. Consistent with this result, ex vivo experiments demonstrated that OT caused renal artery relaxation. Blockade of OT receptors (OXTR) reduced these responses to OT, indicating a direct effect of this peptide on OXTR on this artery. Hypertonic saline (3 M NaCl, 1.8 ml • kg⁻¹ b.wt., i.v.) was infused over 60 s. In sham rats (n = 6), hypertonic saline induced renal vasodilation. The OXTR antagonist (AT; atosiban, 40 µg • kg⁻¹ • h⁻¹, i.v.; n = 7) and renal denervation (RX) reduced the renal vasodilation induced by hypernatremia. The combination of atosiban and renal denervation (RX+AT; n = 7) completely abolished the renal vasodilation induced by sodium overload. Intact rats excreted 51% of the injected sodium within 90 min. Natriuresis was slightly blunted by atosiban and renal denervation (42% and 39% of load, respectively), whereas atosiban with renal denervation reduced sodium excretion to 16% of the load. These results suggest that OT and renal nerves are involved in renal vasodilation and natriuresis induced by acute plasma hypernatremia.     

Regular Cocaine Use Is Associated with Increased Systolic Blood Pressure,Aortic Stiffness and Left Ventricular Mass in Young Otherwise Healthy Individuals

Background

The cardiovascular impact of cocaine use in otherwise healthy individuals who consider themselves ‘social’ users is not well established.

Methods/Results

Twenty regular cocaine users and 20 control subjects were recruited by word-of-mouth. Cardiovascular magnetic resonance was performed to assess cardiac and vascular structure and function. Cocaine users had higher systolic blood pressure compared to non-users (134±11 vs 126±11 mmHg, p = 0.036), a finding independent of age, body surface area, smoking and alcohol consumption. Cocaine use was associated with increased arterial stiffness - reflected by reduced aortic compliance (1.3±0.2 vs 1.7±0.5 cm²×10⁻².mmHg⁻¹, p = 0.004), decreased distensibility (3.8±0.9 vs 5.1±1.4 mmHg⁻¹.10⁻³, p = 0.001), increased stiffness index (2.6±0.6 vs 2.1±0.6, p = 0.005), and higher pulse wave velocity (5.1±0.6 vs 4.4±0.6 m.s⁻¹, p = 0.001). This change in aortic stiffness was independent of vessel wall thickness. Left ventricular mass was 18% higher in cocaine users (124±25 vs 105±16 g, p = 0.01), a finding that was independent of body surface area, and left atrial diameter was larger in the user group than controls (3.8±0.6 vs 3.5±0.3 cm, p = 0.04). The increased left ventricular mass, systolic blood pressure and vascular stiffness measures were all associated with duration and/or frequency of cocaine use. No late gadolinium enhancement or segmental wall motion abnormalities were seen in any of the subjects.

Conclusions

Compared with the non-user control cohort, cocaine users had increased aortic stiffness and systolic blood pressure, associated with greater left ventricular mass. These measures are all well known risk factors for premature cardiovascular events, highlighting the dangers of cocaine use, even in a ‘social’ setting, and have important public health implications.     

TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

Background

RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.

Methods and Results

TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.

Conclusion

RP105 is a potent inhibitor of post-interventional neointima formation.     

Non-Invasive Detection of Coronary Endothelial Response to Sequential Handgrip Exercise in Coronary Artery Disease Patients and Healthy Adults

Objectives

Our objective is to test the hypothesis that coronary endothelial function (CorEndoFx) does not change with repeated isometric handgrip (IHG) stress in CAD patients or healthy subjects.

Background

Coronary responses to endothelial-dependent stressors are important measures of vascular risk that can change in response to environmental stimuli or pharmacologic interventions. The evaluation of the effect of an acute intervention on endothelial response is only valid if the measurement does not change significantly in the short term under normal conditions. Using 3.0 Tesla (T) MRI, we non-invasively compared two coronary artery endothelial function measurements separated by a ten minute interval in healthy subjects and patients with coronary artery disease (CAD).

Methods

Twenty healthy adult subjects and 12 CAD patients were studied on a commercial 3.0 T whole-body MR imaging system. Coronary cross-sectional area (CSA), peak diastolic coronary flow velocity (PDFV) and blood-flow were quantified before and during continuous IHG stress, an endothelial-dependent stressor. The IHG exercise with imaging was repeated after a 10 minute recovery period.

Results

In healthy adults, coronary artery CSA changes and blood-flow increases did not differ between the first and second stresses (mean % change ±SEM, first vs. second stress CSA: 14.8%±3.3% vs. 17.8%±3.6%, p = 0.24; PDFV: 27.5%±4.9% vs. 24.2%±4.5%, p = 0.54; blood-flow: 44.3%±8.3 vs. 44.8%±8.1, p = 0.84). The coronary vasoreactive responses in the CAD patients also did not differ between the first and second stresses (mean % change ±SEM, first stress vs. second stress: CSA: −6.4%±2.0% vs. −5.0%±2.4%, p = 0.22; PDFV: −4.0%±4.6% vs. −4.2%±5.3%, p = 0.83; blood-flow: −9.7%±5.1% vs. −8.7%±6.3%, p = 0.38).

Conclusion

MRI measures of CorEndoFx are unchanged during repeated isometric handgrip exercise tests in CAD patients and healthy adults. These findings demonstrate the repeatability of noninvasive 3T MRI assessment of CorEndoFx and support its use in future studies designed to determine the effects of acute interventions on coronary vasoreactivity.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.     

Sprint Interval and Sprint Continuous Training Increases Circulating CD34+ Cells and Cardio-Respiratory Fitness in Young Healthy Women

Introduction

The improvement of vascular health in the exercising limb can be attained by sprint interval training (SIT). However, the effects on systemic vascular function and on circulating angiogenic cells (CACs) which may contribute to endothelial repair have not been investigated. Additionally, a comparison between SIT and sprint continuous training (SCT) which is less time committing has not been made.

Methods

12 women (22±2 yrs) completed 12 sessions of either SIT (n = 6) or work-matched SCT (n = 6) on 3 days/week. Pre and post-training assessments included brachial artery endothelial function and peripheral blood analysis for CAC number (CD34⁺/CD34⁺CD45^dim). CAC function was measured by migration and adhesion assays. Cardio-respiratory fitness, carotid arterial stiffness and carotid-radial and brachial-foot pulse wave velocity (PWV) were also evaluated.

Results

CD34⁺ CACs increased following training in both groups but CD34⁺CD45^dim did not (Pre CD34⁺: 40±21/10⁵ leukocytes, Post CD34⁺: 56±24/10⁵ leukocytes, main time effect p<0.05). Brachial artery flow-mediated dilation (FMD) increased following SIT but SCT had no effect (Pre SIT: 5.0±3.4%, Post SIT: 5.9±3.0%, Pre SCT: 7.2±2.7%, Post SCT: 6.5±2.9%; group x time interaction p = 0.08). increased in both training groups (Pre: 34.6±4.6 ml•kg•ml⁻¹, Post: 36.9±5.4 ml•kg•ml⁻¹, main time effect p<0.05). CAC function, carotid arterial stiffness and PWV did not change after training (p>0.05).

Discussion

SCT involving little time commitment is comparable to SIT in increasing CD34⁺ cell number and . An increased mobilisation of CD34⁺ CACs suggests that sprint training may be an effective method to enhance vascular repair.     

Pulmonary and Cardiac Function in Asymptomatic Obese Subjects and Changes following a Structured Weight Reduction Program: A Prospective Observational Study

Background

The prevalence of obesity is rising. Obesity can lead to cardiovascular and ventilatory complications through multiple mechanisms. Cardiac and pulmonary function in asymptomatic subjects and the effect of structured dietary programs on cardiac and pulmonary function is unclear.

Objective

To determine lung and cardiac function in asymptomatic obese adults and to evaluate whether weight loss positively affects functional parameters.

Methods

We prospectively evaluated bodyplethysmographic and echocardiographic data in asymptomatic subjects undergoing a structured one-year weight reduction program.

Results

74 subjects (32 male, 42 female; mean age 42±12 years) with an average BMI 42.5±7.9, body weight 123.7±24.9 kg were enrolled. Body weight correlated negatively with vital capacity (R = −0.42, p<0.001), FEV1 (R = −0.497, p<0.001) and positively with P 0.1 (R = 0.32, p = 0.02) and myocardial mass (R = 0.419, p = 0.002). After 4 months the study subjects had significantly reduced their body weight (−26.0±11.8 kg) and BMI (−8.9±3.8) associated with a significant improvement of lung function (absolute changes: vital capacity +5.5±7.5% pred., p<0.001; FEV1+9.8±8.3% pred., p<0.001, ITGV+16.4±16.0% pred., p<0.001, SR tot −17.4±41.5% pred., p<0.01). Moreover, P0.1/Pimax decreased to 47.7% (p<0.01) indicating a decreased respiratory load. The change of FEV1 correlated significantly with the change of body weight (R = −0.31, p = 0.03). Echocardiography demonstrated reduced myocardial wall thickness (−0.08±0.2 cm, p = 0.02) and improved left ventricular myocardial performance index (−0.16±0.35, p = 0.02). Mitral annular plane systolic excursion (+0.14, p = 0.03) and pulmonary outflow acceleration time (AT +26.65±41.3 ms, p = 0.001) increased.

Conclusion

Even in asymptomatic individuals obesity is associated with abnormalities in pulmonary and cardiac function and increased myocardial mass. All the abnormalities can be reversed by a weight reduction program.     

Central Nervous System PET-CT Imaging Reveals Regional Impairments in Pediatric Patients with Wolfram Syndrome

Wolfram syndrome (WFS) is inherited as an autosomal recessive disease with main clinical features of diabetes mellitus, optic atrophy, diabetes insipidus and deafness. However, various neurological defects may also be detected. The aim of this study was to evaluate aspects of brain structure and function using PET-CT (positron emission tomography and computed tomography) and MRI (magnetic resonance imaging) in pediatric patients with WFS. Regional changes in brain glucose metabolism were measured using standardized uptake values (SUVs) based on images of (¹⁸F) fluorodeoxyglucose (FDG) uptake in 7 WFS patients aged 10.1–16.0 years (mean 12.9±2.4) and in 20 healthy children aged 3–17.9 years (mean 12.8±4.1). In all patients the diagnosis of WFS was confirmed by DNA sequencing of the WFS1 gene. Hierarchical clustering showed remarkable similarities of glucose uptake patterns among WFS patients and their differences from the control group. SUV data were subsequently standardized for age groups <13 years old and>13 years old to account for developmental differences. Reduced SUVs in WFS patients as compared to the control group for the bilateral brain regions such as occipital lobe (−1.24±1.20 vs. −0.13±1.05; p = 0.028) and cerebellum (−1.11±0.69 vs. −0.204±1.00; p = 0.036) were observed and the same tendency for cingulate (−1.13±1.05 vs. −0.15±1.12; p = 0.056), temporal lobe (−1.10±0.98 vs. −0.15±1.10; p = 0.057), parietal lobe (−1.06±1.20 vs. −0.08±1.08; p = 0.058), central region (−1.01±1.04 vs. −0.09±1.06; p = 0.060), basal ganglia (−1.05±0.74 vs. −0.20±1.07; p = 0.066) and mesial temporal lobe (−1.06±0.82 vs. −0.26±1.08; p = 0.087) was also noticed. After adjusting for multiple hypothesis testing, the differences in glucose uptake were non-significant. For the first time, regional differences in brain glucose metabolism among patients with WFS were shown using PET-CT imaging.     

Heart on a Plate: Histological and Functional Assessment of Isolated Adult Zebrafish Hearts Maintained in Culture

The zebrafish is increasingly used for cardiovascular genetic and functional studies. We present a novel protocol to maintain and monitor whole isolated beating adult zebrafish hearts in culture for long-term experiments. Excised whole adult zebrafish hearts were transferred directly into culture dishes containing optimized L-15 Leibovitz growth medium and maintained for 5 days. Hearts were assessed daily using video-edge analysis of ventricle function using low power microscopy images. High-throughput histology techniques were used to assess changes in myocardial architecture and cell viability. Mean spontaneous Heart rate (HR, min⁻¹) declined significantly between day 0 and day 1 in culture (96.7±19.5 to 45.2±8.2 min⁻¹, mean±SD, p = 0.001), and thereafter declined more slowly to 27.6±7.2 min⁻¹ on day 5. Ventricle wall motion amplitude (WMA) did not change until day 4 in culture (day 0, 46.7±13.0 µm vs day 4, 16.9±1.9 µm, p = 0.08). Contraction velocity (CV) declined between day 0 and day 3 (35.6±14.8 vs 15.2±5.3 µms⁻¹, respectively, p = 0.012) while relaxation velocity (RV) declined quite rapidly (day 0, 72.5±11.9 vs day 1, 29.5±5.8 µms⁻¹, p = 0.03). HR and WMA responded consistently to isoproterenol from day 0 to day 5 in culture while CV and RV showed less consistent responses to beta-agonist. Cellular architecture and cross-striation pattern of cardiomyocytes remained unchanged up to day 3 in culture and thereafter showed significant deterioration with loss of striation pattern, pyknotic nuclei and cell swelling. Apoptotic markers within the myocardium became increasingly frequent by day 3 in culture. Whole adult zebrafish hearts can be maintained in culture-medium for up to 3 days. However, after day-3 there is significant deterioration in ventricle function and heart rate accompanied by significant histological changes consistent with cell death and loss of cardiomyocyte cell integrity. Further studies are needed to assess whether this preparation can be optimised for longer term survival.     

Alterations of Neuromuscular Function after the World's Most Challenging Mountain Ultra-Marathon

We investigated the physiological consequences of the most challenging mountain ultra-marathon (MUM) in the world: a 330-km trail run with 24000 m of positive and negative elevation change. Neuromuscular fatigue (NMF) was assessed before (Pre-), during (Mid-) and after (Post-) the MUM in experienced ultra-marathon runners (n = 15; finish time  = 122.43 hours ±17.21 hours) and in Pre- and Post- in a control group with a similar level of sleep deprivation (n = 8). Blood markers of muscle inflammation and damage were analyzed at Pre- and Post-. Mean ± SD maximal voluntary contraction force declined significantly at Mid- (−13±17% and −10±16%, P<0.05 for knee extensor, KE, and plantar flexor muscles, PF, respectively), and further decreased at Post- (−24±13% and −26±19%, P<0.01) with alteration of the central activation ratio (−24±24% and −28±34% between Pre- and Post-, P<0.05) in runners whereas these parameters did not change in the control group. Peripheral NMF markers such as 100 Hz doublet (KE: −18±18% and PF: −20±15%, P<0.01) and peak twitch (KE: −33±12%, P<0.001 and PF: −19±14%, P<0.01) were also altered in runners but not in controls. Post-MUM blood concentrations of creatine kinase (3719±3045 Ul·¹), lactate dehydrogenase (1145±511 UI·L⁻¹), C-Reactive Protein (13.1±7.5 mg·L⁻¹) and myoglobin (449.3±338.2 µg·L⁻¹) were higher (P<0.001) than at Pre- in runners but not in controls. Our findings revealed less neuromuscular fatigue, muscle damage and inflammation than in shorter MUMs. In conclusion, paradoxically, such extreme exercise seems to induce a relative muscle preservation process due likely to a protective anticipatory pacing strategy during the first half of MUM and sleep deprivation in the second half.     

Impact of Gout on Left Atrial Function: A Prospective Speckle-Tracking Echocardiographic Study

The purpose of our study was to evaluate the left ventricular (LV) and left atrial (LA) function in patients with gout. A total of 173 patients underwent a comprehensive Doppler-echocardiography examination. Participants were divided into four groups–Stage 0: control (n = 35), Stage I: asymptomatic hyperuricemia (n = 30), Stage II: gouty arthritis without tophi (n = 58), and Stage III: tophaceous gout (n = 50). Serum uric acid levels were not significantly different between stage I, II and III. Stage III patients demonstrated a higher ratio of the transmitral and myocardial peak early diastolic velocities (E/Em) (10.50±3.18 vs. 8.58±2.07; P = 0.008), and larger maximal LA volume index (LAVi) (29.60±9.89 vs. 20.07±4.76 ml/m²; P<0.001) compared with controls. Stage III patients had decreased LV global longitudinal systolic strain (LVε) compared with controls (−20.2±3.06 vs. −21.79±2.27; P = 0.002). Stage III patients also had decreased peak atrial longitudinal strain rate during ventricular systole (ALSR_syst), peak atrial longitudinal strain rate during ventricular early diastole (ALSR_early), and peak atrial longitudinal strain rate during ventricular late diastole (ALSR_late) compared with controls (1.73±0.48 vs. 2.05±0.55 1/s, −1.44±0.53 vs. −2.07±0.84 1/s, −2.07±0.7 vs. −2.66±0.91 1/s, respectively; all P<0.005). Multiple regression analysis revealed severity of gout had an independent negative impact on LA pump function (ALSR_late). In conclusion, gout caused LV diastolic dysfunction, LV subclinical systolic dysfunction and LA reservoir, conduit, and booster pump dysfunction.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Chronic Aerobic Exercise Associated to Dietary Modification Improve Endothelial Function and eNOS Expression in High Fat Fed Hamsters

Obesity is epidemic in the western world and central adipose tissue deposition points to increased cardiovascular morbidity and mortality, independently of any association between obesity and other cardiovascular risk factors. Physical exercise has been used as non-pharmacological treatment to significantly reverse/attenuate obesity comorbidities. In this study we have investigated effects of exercise and/or dietary modification on microcirculatory function, body composition, serum glucose, iNOS and eNOS expression on 120 male hamsters treated for 12 weeks with high fat chow (HF, n = 30) starting on the 21^st day of birth. From week 12 to 20, animals were randomly separated in HF (no treatment change), return to standard chow (HFSC, n = 30), high fat chow associated to an aerobic exercise training program (AET) (HFEX, n = 30) and return to standard chow+AET (HFSCEX, n = 30). Microvascular reactivity in response to acetylcholine and sodium nitroprusside and macromolecular permeability increase induced by 30 minutes ischemia followed by reperfusion were assessed on the cheek pouch preparation. Total body fat and aorta eNOS and iNOS expression by immunoblotting assay were evaluated on the experimental day. Compared to HFSC and HFSCEX groups, HF and HFEX ones presented increased visceral fat [(mean±SEM) (HF)4.9±1.5 g and (HFEX)4.7±0.9 g vs. (HFSC)*3.0±0.7 g and (HFSCEX)*1.9±0.4 g/100 g BW]; impaired endothelial-dependent vasodilatation [Ach 10⁻⁸ M (HF)87.9±2.7%; (HFSC)*116.7±5.9%; (HFEX)*109.1±4.6%; (HFSCEX)*105±2.8%; Ach10⁻⁶ M (HF)95.3±3.1%; (HFSC)*126±6.2%; (HFEX)*122.5±2.8%; (HFSCEX)*118.1±4.3% and Ach10⁻⁴ M (HF)109.5±4.8%; (HFSC)*149.6±6.6%; (HFEX)*143.5±5.4% and (HFSCEX)*139.4±5.2%], macromolecular permeability increase after ischemia/reperfusion [(HF)40.5±4.2; (HFSC)*19.0±1.6; (HFEX)*18.6±2.1 and (HFSCEX)* 21.5±3.7 leaks/cm²), decreased eNOS expression, increased leptin and glycaemic levels. Endothelial-independent microvascular reactivity was similar between groups, suggesting that only endothelial damage had occurred. Our results indicate that an aerobic routine and/or dietary modification may cause significant improvements to high fat fed animals, diminishing visceral depots, increasing eNOS expression and reducing microcirculatory dysfunction.     

Electrocardiogram-Gated 320-Slice Multidetector Computed Tomography for the Measurement of Pulmonary Arterial Distensibility in Chronic Thromboembolic Pulmonary Hypertension

Background

We aimed to study whether pulmonary arterial distensibility (PAD) correlates with hemodynamic parameters in chronic thromboembolic pulmonary hypertension (CTEPH) using electrocardiogram (ECG)-gated 320-slice multidetector computed tomography (MDCT).

Methods and Findings

ECG-gated 320-slice MDCT and right heart catheterization (RHC) was performed in 53 subjects (60.6±11.4 years old; 37 females) with CTEPH. We retrospectively measured the minimum and maximum values of the cross sectional area (CSA) of the main pulmonary artery (mainPA), right pulmonary artery (rtPA), and left pulmonary artery (ltPA) during one heartbeat. PAD was calculated using the following formula: PAD = [(CSAmaximum−CSAminimum)/CSAmaximum]×100(%). The correlation between hemodynamic parameters and PAD was assessed. Mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR) were 40.8±8.7 mmHg and 8.3±3.0 wood units, respectively. PAD values were as follows: mainPA (14.0±5.0%), rtPA (12.8±5.6%), and ltPA (9.7±4.6%). Good correlations existed between mainPAD, with mPAP (r = −0.594, p<0.001) and PVR (r = −0.659, p<0.001). The correlation coefficients between rtPAD and ltPAD with pulmonary hemodynamics were all lower or equal than for mainPAD.

Conclusions

PAD measured using ECG-gated 320-slice MDCT correlates with pulmonary hemodynamics in subjects with CTEPH. The mainPA is suitable for PAD measurement.     

Daily Energy Expenditure,Cardiorespiratory Fitness and Glycaemic Control in People with Type 1 Diabetes

Objective

Encouraging daily physical activity improves cardiorespiratory fitness and many cardiovascular risk factors. However, increasing physical activity often creates a challenge for people with type 1 diabetes, because of difficulties maintaining euglycemia in the face of altered food intake and adjustments to insulin doses. Our aim was to examine the triangular relationship between glucose control measured by continuous glucose monitoring system (CGMS), objective measures of total daily energy expenditure (TEE) recorded by a multi-sensory monitoring device, and cardiorespiratory fitness (CRF), in free-living subjects with type 1 diabetes.

Research Design and Methods

Twenty-three individuals (12 women) with type 1 diabetes who were free from micro- and macrovascular complications were recruited. TEE and glucose control were monitored simultaneously for up to 12 days, using a multi-sensory device and CGMS respectively. CRF was recorded as V02 max from a maximal treadmill test with the Bruce protocol.

Results

Subjects (mean±SD) were aged 37±11 years, with BMI = 26.5±5.1 kg.m⁻², HbA_1c = 7.7±1.3% (61±14 mmol/mol) and V02 max (ml.min⁻¹.kg⁻¹)  = 39.9±8.4 (range 22.4 – 58.6). TEE (36.3±5.5 kcal.kg⁻¹.day⁻¹) was strongly associated with CRF(39.9±8.4 ml.min⁻¹.kg⁻¹) independently of sex (r = 0.63, p<0.01). However, neither TEE (r = −0.20, p = 0.36) nor CRF (r = −0.20, p = 0.39; adjusted for sex), were significantly associated with mean glycaemia measured by CGMS.

Conclusion

Higher levels of energy expenditure (due to a more active lifestyle) are associated with increased cardiorespiratory fitness, but not necessarily better glycaemic control. Since increased levels of energy expenditure and good glycaemic control are both needed to protect against diabetes-related complications our data suggest they need to be achieved independently.     

Whole-Body Insulin Sensitivity Rather than Body-Mass-Index Determines Fasting and Post-Glucose-Load Growth Hormone Concentrations

Background

Obese, non-acromegalic persons show lower growth hormone (GH) concentrations at fasting and reduced GH nadir during an oral glucose tolerance test (OGTT). However, this finding has never been studied with regard to whole-body insulin-sensitivity as a possible regulator.

Methods

In this retrospective analysis, non-acromegalic (NonACRO, n = 161) and acromegalic (ACRO, n = 35), non-diabetic subjects were subdivided into insulin-sensitive (IS) and –resistant (IR) groups according to the Clamp-like Index (CLIX)-threshold of 5 mg·kg⁻¹·min⁻¹ from the OGTT.

Results

Non-acromegalic IS (CLIX: 8.8±0.4 mg·kg⁻¹·min⁻¹) persons with similar age and sex distribution, but lower (p<0.001) body-mass-index (BMI = 25±0 kg/m², 84% females, 56±1 years) had 59% and 70%, respectively, higher (p<0.03) fasting GH and OGTT GH area under the curve concentrations than IR (CLIX: 3.5±0.1 mg·kg⁻¹·min⁻¹, p<0.001) subjects (BMI = 29±1 kg/m², 73% females, 58±1 years). When comparing on average overweight non-acromegalic IS and IR with similar anthropometry (IS: BMI: 27±0 kg/m², 82% females, 58±2 years; IR: BMI: 27±0 kg/m², 71% females, 60±1 years), but different CLIX (IS: 8.7±0.9 vs. IR: 3.8±0.1 mg·kg⁻¹·min⁻¹, p<0.001), the results remained almost the same. In addition, when adjusted for OGTT-mediated glucose rise, GH fall was less pronounced in IR. In contrast, in acromegalic subjects, no difference was found between IS and IR patients with regard to fasting and post-glucose-load GH concentrations.

Conclusions

Circulating GH concentrations at fasting and during the OGTT are lower in non-acromegalic insulin-resistant subjects. This study seems the first to demonstrate that insulin sensitivity rather than body-mass modulates fasting and post-glucose-load GH concentrations in non-diabetic non–acromegalic subjects.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Assessment of Metabolomic and Proteomic Biomarkers in Detection and Prognosis of Progression of Renal Function in Chronic Kidney Disease

Chronic kidney disease (CKD) is part of a number of systemic and renal diseases and may reach epidemic proportions over the next decade. Efforts have been made to improve diagnosis and management of CKD. We hypothesised that combining metabolomic and proteomic approaches could generate a more systemic and complete view of the disease mechanisms. To test this approach, we examined samples from a cohort of 49 patients representing different stages of CKD. Urine samples were analysed for proteomic changes using capillary electrophoresis-mass spectrometry and urine and plasma samples for metabolomic changes using different mass spectrometry-based techniques. The training set included 20 CKD patients selected according to their estimated glomerular filtration rate (eGFR) at mild (59.9±16.5 mL/min/1.73 m²; n = 10) or advanced (8.9±4.5 mL/min/1.73 m²; n = 10) CKD and the remaining 29 patients left for the test set. We identified a panel of 76 statistically significant metabolites and peptides that correlated with CKD in the training set. We combined these biomarkers in different classifiers and then performed correlation analyses with eGFR at baseline and follow-up after 2.8±0.8 years in the test set. A solely plasma metabolite biomarker-based classifier significantly correlated with the loss of kidney function in the test set at baseline and follow-up (ρ = −0.8031; p<0.0001 and ρ = −0.6009; p = 0.0019, respectively). Similarly, a urinary metabolite biomarker-based classifier did reveal significant association to kidney function (ρ = −0.6557; p = 0.0001 and ρ = −0.6574; p = 0.0005). A classifier utilising 46 identified urinary peptide biomarkers performed statistically equivalent to the urinary and plasma metabolite classifier (ρ = −0.7752; p<0.0001 and ρ = −0.8400; p<0.0001). The combination of both urinary proteomic and urinary and plasma metabolic biomarkers did not improve the correlation with eGFR. In conclusion, we found excellent association of plasma and urinary metabolites and urinary peptides with kidney function, and disease progression, but no added value in combining the different biomarkers data.     

The Osteoblastogenesis Potential of Adipose Mesenchymal Stem Cells in Myeloma Patients Who Had Received Intensive Therapy

Multiple myeloma (MM) is characterized by advanced osteolytic lesions resulting from the activation of osteoclasts (OCs) and inhibition of osteoblasts (OBs). OBs are derived from mesenchymal stem cells (MSCs) from the bone marrow (BM), however the pool and function of BMMSCs in MM patients (MM-BMMSCs) are reduced by myeloma cells (MCs) and cytokines secreted from MCs and related anti-MM treatment. Such reduction in MM-BMMSCs currently cannot be restored by any means. Recently, genetic aberrations of MM-BMMSCs have been noted, which further impaired their differentiation toward OBs. We hypothesize that the MSCs derived from adipose tissue (ADMSCs) can be used as alternative MSC sources to enhance the pool and function of OBs. Therefore, the purpose of this study was to compare the osteogenesis ability of paired ADMSCs and BMMSCs in MM patients who had completed intensive therapy. Fifteen MM patients who had received bortezomib-based induction and autologous transplantation were enrolled. At the third month after the transplant, the paired ADMSCs and BMMSCs were obtained and cultured. Compared with the BMMSCs, the ADMSCs exhibited a significantly higher expansion capacity (100% vs 13%, respectively; P = .001) and shorter doubling time (28 hours vs 115 hours, respectively; P = .019). After inducing osteogenic differentiation, although the ALP activity did not differ between the ADMSCs and BMMSCs (0.78 U/µg vs 0.74±0.14 U/µg, respectively; P = .834), the ADMSCs still exhibited higher calcium mineralization, which was determined using Alizarin red S (1029 nmole vs 341 nmole, respectively; P = .001) and von Kossa staining (2.6 E+05 µm² vs 5 E+04 µm², respectively; P = .042), than the BMMSCs did. Our results suggested that ADMSCs are a feasible MSC source for enhancing the pool and function of OBs in MM patients who have received intensive therapy.