Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO(2)max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Prolonged amelioration of acute lung allograft rejection by overexpression of human interleukin-10 under control of a long acting ubiquitin C promoter in rats

BACKGROUND: The prolonged effect of electroporation-mediated human interleukin-10 (hIL-10) overexpression in skeletal muscle under the control of the constitutional polyubiquitin C promoter (pUb hIL-10) on rat lung allograft rejection was evaluated. METHODS: Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Either 2.5 mug pCIK hIL-10 (hIL-10/cytomegalovirus early promoter enhancer) alone (Group I/sacrifice Day 5 and II/sacrifice Day 10) or in combination with 2.5 mug pUb hIL-10 (hIL-10/UbC promoter; Group III/sacrifice Day 10) were injected into the tibialis anterior muscle of the recipient, followed by electroporation 24 hours before transplantation. Animals in Control Groups IV and V without gene transfer were euthanized on Day 5 and 10, respectively. All animals received a daily non-therapeutic dose of cyclosporine A (2.5 mg/kg). RESULTS: In Control Group IV, complete rejection (median A3B3) was noted on Day 5 with a Pao(2) of 43 +/- 9 mm Hg. In recipients of Control Group V, measurement of gas exchange on Day 10 and rejection grading was impossible because of complete destruction of the allograft. Group I animals on Day 5 (233 +/- 123 mm Hg; p = 0.02 vs Group IV) and Group II animals on Day 10 (150 +/- 139 mm Hg; p = 0.15 vs Group IV) demonstrated improved graft function. Graft function in Group III was further improved on Day 10 (299 +/- 123 mm Hg; p = 0.002 vs Group IV; p = 0.05 vs Group II; p = 0.36 vs Group I). Rejection was significantly reduced in Group III (median, A2B2) compared with Group II (median, A4B3; p < 0.05). CONCLUSIONS: Interleukin-10 overexpression under control of the constitutive ubiquitin C promoter ameliorates acute rejection and preserves lung graft function for a prolonged time.

Diastolic dysfunction in human cardiac allografts is related with reduced SERCA2a gene expression

Previous studies demonstrated that impaired left ventricular (LV) relaxation in cardiac allografts limits exercise tolerance post-transplant despite preserved systolic ejection fraction (EF). This study tested in human cardiac allografts whether the isovolumic relaxation time (IVRT), which provides the basis for most of diastolic LV filling, relates with gene expression of regulatory proteins of calcium homeostasis or cardiac matrix proteins. Gene expression was studied in 31 heart transplant recipients (25 male, 6 female) 13-83 months post-transplant with LVEF >50%, LV end-diastolic pressure <20 mmHg, normal LV mass index and without allograft rejection or significant cardiac pathology. IVRT related with the other diastolic parameters e-wave velocity (r = -0.46; p = 0.01), e/a-wave ratio (r = -0.5; p < 0.01) but not with heart frequency (r = -0.16; p = 0.4). No relation of IVRT was observed for immunosuppression, mean rejection grade or other medication. IVRT was not related with gene expression of desmin, collagen I, phospholamban, the Na+-Ca2+ exchanger, the ryanodine receptor or interstitial fibrosis but correlated inversely with SERCA2a (r = -0.48; p = 0.02). Prolonged IVRT is associated with decreased SERCA2a expression in cardiac allografts without significant other pathology. Similar observations in non-transplanted patients with diastolic failure suggest that decreased SERCA2a expression is an important common pathomechanism.

CRF receptors in the rodent and human cardiovascular systems: Species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine or [(125)I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF(2) receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF(2) receptors were detected with (125)I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF(2) receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.

Reduced atrial connexin43 expression after pediatric heart surgery

Myocardial dysfunction and arrhythmias may be induced by congenital heart defects, but also be the result of heart surgery with cardiopulmonary bypass (CPB), potentially caused by differential expression of connexin40 (Cx40) and connexin43 (Cx43). In 16 pediatric patients undergoing corrective heart surgery, connexin mRNA expression was studied in volume overloaded (VO group, n=8) and not overloaded (NO group, n=8) right atrial myocardium, excised before and after CPB. Additionally, in eight of these patients ventricular specimens were investigated. The atrial Cx43 expression decreased during CPB, which was restricted to the VO group (p=0.008). In contrast, atrial Cx40 mRNA did not change during CPB. In ventricular myocardium compared to atrial mRNA levels, Cx40 was lower (p=0.006) and Cx43 higher (p=0.017) expressed, without significant change during CPB. This study revealed a significant influence of CPB and the underlying heart defect on Cx43 expression.

Absence of histamine-induced nitric oxide release in the human radial artery: implications for vasospasm of coronary artery bypass vessels

Radial artery (RA) bypass grafts can develop severe vasospasm. As histamine is known to induce vasospasm, its effect on RA was assessed compared with the classic bypass vessels internal mammary artery (MA) and saphenous vein (SV). The vessels were examined in organ chambers for isometric tension recording. Histamine induced contractions on baseline; the sensitivity was higher in RA and SV than MA. After precontraction with norepinephrine, histamine did not evoke relaxations of RA but induced relaxations of MA and less of SV at lower concentrations; it induced contractions at higher concentrations, reaching similar levels in all three vessels. Indomethacin did not affect the response of MA and RA but potentiated relaxations and reduced contractions of SV. Endothelium removal, N(omega)-nitro-L-arginine methyl ester (L-NAME), or the H2-receptor blocker cimetidine did not affect the response of RA, but inhibited relaxations and enhanced contractions in MA and inhibited relaxations in SV; in the latter, only L-NAME enhanced contractions. Real-time PCR detected much lower expression of endothelial H2-receptor in RA than MA or SV. Western blots revealed similar endothelial nitric oxide (NO) synthase expression in all three vessels. Relaxations to acetylcholine were identical in RA and MA. Thus histamine releases NO by activating the endothelial H2-receptor, the expression of which is much lower in RA than MA or SV. H2-receptor activation also releases prostaglandins in SV, partially antagonizing NO. The lack of histamine-induced NO production represents a possible mechanism of RA vasospasm.

Two-step postmortem angiography with a modified heart-lung machine: preliminary results

OBJECTIVE: The purpose of this study was to adapt and improve a minimally invasive two-step postmortem angiographic technique for use on human cadavers. Detailed mapping of the entire vascular system is almost impossible with conventional autopsy tools. The technique described should be valuable in the diagnosis of vascular abnormalities. MATERIALS AND METHODS: Postmortem perfusion with an oily liquid is established with a circulation machine. An oily contrast agent is introduced as a bolus injection, and radiographic imaging is performed. In this pilot study, the upper or lower extremities of four human cadavers were perfused. In two cases, the vascular system of a lower extremity was visualized with anterograde perfusion of the arteries. In the other two cases, in which the suspected cause of death was drug intoxication, the veins of an upper extremity were visualized with retrograde perfusion of the venous system. RESULTS: In each case, the vascular system was visualized up to the level of the small supplying and draining vessels. In three of the four cases, vascular abnormalities were found. In one instance, a venous injection mark engendered by the self-administration of drugs was rendered visible by exudation of the contrast agent. In the other two cases, occlusion of the arteries and veins was apparent. CONCLUSION: The method described is readily applicable to human cadavers. After establishment of postmortem perfusion with paraffin oil and injection of the oily contrast agent, the vascular system can be investigated in detail and vascular abnormalities rendered visible.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Microvascular response to metabolic and pressure challenge in the human coronary circulation

In vivo observations of microcirculatory behavior during autoregulation and adaptation to varying myocardial oxygen demand are scarce in the human coronary system. This study assessed microvascular reactions to controlled metabolic and pressure provocation [bicycle exercise and external counterpulsation (ECP)]. In 20 healthy subjects, quantitative myocardial contrast echocardiography and arterial applanation tonometry were performed during increasing ECP levels, as well as before and during bicycle exercise. Myocardial blood flow (MBF; ml·min(-1)·g(-1)), the relative blood volume (rBV; ml/ml), the coronary vascular resistance index (CVRI; dyn·s·cm(-5)/g), the pressure-work index (PWI), and the pressure-rate product (mmHg/min) were assessed. MBF remained unchanged during ECP (1.08 ± 0.44 at baseline to 0.92 ± 0.38 at high-level ECP). Bicycle exercise led to an increase in MBF from 1.03 ± 0.39 to 3.42 ± 1.11 (P < 0.001). The rBV remained unchanged during ECP, whereas it increased under exercise from 0.13 ± 0.033 to 0.22 ± 0.07 (P < 0.001). The CVRI showed a marked increase under ECP from 7.40 ± 3.38 to 11.05 ± 5.43 and significantly dropped under exercise from 7.40 ± 2.78 to 2.21 ± 0.87 (both P < 0.001). There was a significant correlation between PWI and MBF in the pooled exercise data (slope: +0.162). During ECP, the relationship remained similar (slope: +0.153). Whereas physical exercise decreases coronary vascular resistance and induces considerable functional capillary recruitment, diastolic pressure transients up to 140 mmHg trigger arteriolar vasoconstriction, keeping MBF and functional capillary density constant. Demand-supply matching was maintained over the entire ECP pressure range.

Reciprocal relationship between left ventricular filling pressure and the recruitable human coronary collateral circulation

AIMS The aim of our study in patients with coronary artery disease (CAD) and present, or absent, myocardial ischaemia during coronary occlusion was to test whether (i) left ventricular (LV) filling pressure is influenced by the collateral circulation and, on the other hand, that (ii) its resistance to flow is directly associated with LV filling pressure. METHODS AND RESULTS In 50 patients with CAD, the following parameters were obtained before and during a 60 s balloon occlusion: LV, aortic (Pao) and coronary pressure (Poccl), flow velocity (Voccl), central venous pressure (CVP), and coronary flow velocity after coronary angioplasty (V(Ø-occl)). The following variables were determined and analysed at 10 s intervals during occlusion, and at 60 s of occlusion: LV end-diastolic pressure (LVEDP), velocity-derived (CFIv) and pressure-derived collateral flow index (CFIp), coronary collateral (Rcoll), and peripheral resistance index to flow (Rperiph). Patients with ECG signs of ischaemia during coronary occlusion (insufficient collaterals, n = 33) had higher values of LVEDP over the entire course of occlusion than those without ECG signs of ischaemia during occlusion (sufficient collaterals, n = 17). Despite no ischaemia in the latter, there was an increase in LVEDP from 20 to 60 s of occlusion. In patients with insufficient collaterals, CFIv decreased and CFIp increased during occlusion. Beyond an occlusive LVEDP > 27 mmHg, Rcoll and Rperiph increased as a function of LVEDP. CONCLUSION Recruitable collaterals are reciprocally tied to LV filling pressure during occlusion. If poorly developed, they affect it via myocardial ischaemia; if well grown, LV filling pressure still increases gradually during occlusion despite the absence of ischaemia indicating transmission of collateral perfusion pressure to the LV. With low, but not high, collateral flow, resistance to collateral as well as coronary peripheral flow is related to LV filling pressure in the high range.

NaV1.5 and interacting proteins in human arrhythmogenic cardiomyopathy

Evaluation of: Noorman M, Hakim S, Kessler E et al. Remodeling of the cardiac sodium channel, connexin43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy. Heart Rhythm 10(3), 412-419 (2013). Arrhythmogenic cardiomyopathy (AC) is a heart muscle disease characterized by a progressive replacement of the ventricular myocardium with adipose and fibrous tissue. This disease is often associated with mutations in genes encoding desmosomal proteins in the majority of patients. Based on results obtained from recent experimental models, a disturbed distribution of gap junction proteins and cardiac sodium channels may also be observed in AC phenotypes, secondary to desmosomal dysfunction. The study from Noorman et al. examined heart sections from patients diagnosed with AC and performed immunohistochemical analyses of N-cadherin, PKP2, PKG, Cx43 and the cardiac sodium channel NaV1.5. Altered expression/distribution of Cx43, PKG and NaV1.5 was found in most cases of patients with AC. The altered expression and/or distribution of NaV1.5 channels in AC hearts may play a mechanistic role in the arrhythmias leading to sudden cardiac death in AC patients. Thus, NaV1.5 should be considered as a supplemental element in the evaluation of risk stratification and management strategies. However, additional experiments are required to clearly understand the mechanisms leading to AC phenotypes.

Bodily and Embodied: Being Human in the Tradition of the Hebrew Bible

A depiction of the ancient Hebrew understanding of the human being must take into account the fact that the Bible does not contain a systematic anthropology, but unfolds the multiplicity of human existence inductively, aspectively, and in narrative fashion. In comparison to Greek body/soul dualism, but also in the context of body-(de-)construction and gender debates, this circumstance makes it a treasure trove of interesting, often contrasting recollections and insights with liberating potential. This assertion will be illustrated concretely in terms of the nexus points of the human body (throat, heart, and womb), the relationship of humans to animals and angels, and the questions of the power and value of a human being.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Estimation of heart weight by post-mortem cardiac magnetic resonance imaging

Introduction The aim of this study was to determine which single measurement on post-mortem cardiac MR reflects actual heart weight as measured at autopsy, assess the intra- and inter-observer reliability of MR measurements, derive a formula to predict heart weight from MR measurements and test the accuracy of the formula to prospectively predict heart weight. Materials and methods 53 human cadavers underwent post-mortem cardiac MR and forensic autopsy. In Phase 1, left ventricular area and wall thickness were measured on short axis and four chamber view images of 29 cases. All measurements were correlated to heart weight at autopsy using linear regression analysis. In Phase 2, single left ventricular area measurements on four chamber view images (LVA_4C) from 24 cases were used to predict heart weight at autopsy based on equations derived during Phase 1. Intra-class correlation coefficient (ICC) was used to determine inter- and intra-reader agreement. Results Heart weight strongly correlates with LVA_4C (r=0.78 M; p<0.001). Intra-reader and inter-reader reliability was excellent for LVA_4C (ICC=0.81–0.91; p<0.001 and ICC=0.90; p<0.001 respectively). A simplified formula for heart weight ([g]≈LVA_4C [mm2]×0.11) was derived based on linear regression analysis. Conclusions This study shows that single circumferential area measurements of the left ventricle in the four chamber view on post-mortem cardiac MR reflect actual heart weight as measured at autopsy. These measurements yield an excellent intra- and inter-reader reliability and can be used to predict heart weight prior to autopsy or to give a reasonable estimate of heart weight in cases where autopsy is not performed.