Stephen N. Hunyor

Mesenchymal Stem Cells: Isolation, Characterisation and In Vivo Fluorescent Dye Tracking

Cell therapies have been used to regenerate the heart by direct myocardial delivery, by coronary infusion and by surface attached scaffolds. Multipotent mesenchymal stem cells (MSC) with capacity to differentiate into cardiomyocytes and other cell lines have been predominantly trialled in rodents. However, large animal models are increasingly needed to translate basic research into new, safe regenerative therapies. Understanding the mode of action of cell therapies in the mammalian heart has been limited by cell tracking capability. This study examined the ability to track the fate of allogeneic MSC in sheep using various fluorescent dyes. MSC isolated from sheep bone marrow were grown in culture following extraction and flow cytometric characterisation. After labelling with fluorescent tracking dyes (e.g. CFSE and DiI) cells were tested for in vitro and in vivo signal up to six weeks. Labelling effect on cell division and differentiation was studied. Several dyes lost fluorescence and slowed cell division. However, the thiol reactive dye CM-DiI showed detectable in vivo fluorescence in labelled MSC six weeks after injection into sheep skeletal muscle and two weeks after implantation of an MSC coated biomaterial scaffold. CM-DiI labelled MSC differentiated in vitro showed label retention over four weeks. The fluorescent membrane dye CM-DiI tracks implanted sheep MSC and provides an alternative to traditional cell markers such as gene modified GFP.

β3 Adrenergic Stimulation of the Cardiac Na+-K+ Pump by Reversal of an Inhibitory Oxidative Modification

Background— Inhibition of L-type Ca2+ current contributes to negative inotropy of &bgr;3 adrenergic receptor (&bgr;3 AR) activation, but effects on other determinants of excitation-contraction coupling are not known. Of these, the Na+-K+ pump is of particular interest because of adverse effects attributed to high cardiac myocyte Na+ levels and upregulation of the &bgr;3 AR in heart failure. Methods and Results— We voltage clamped rabbit ventricular myocytes and identified electrogenic Na+-K+ pump current (Ip) as the shift in holding current induced by ouabain. The synthetic &bgr;3 AR agonists BRL37344 and CL316,243 and the natural agonist norepinephrine increased Ip. Pump stimulation was insensitive to the &bgr;1/&bgr;2 AR antagonist nadolol and the protein kinase A inhibitor H-89 but sensitive to the &bgr;3 AR antagonist L-748,337. Blockade of nitric oxide synthase abolished pump stimulation and an increase in fluorescence of myocytes loaded with a nitric oxide–sensitive dye. Exposure of myocytes to &bgr;3 AR agonists decreased &bgr;1 Na+-K+ pump subunit glutathionylation, an oxidative modification that causes pump inhibition. The in vivo relevance of this was indicated by an increase in myocardial &bgr;1 pump subunit glutathionylation with elimination of &bgr;3 AR–mediated signaling in &bgr;3 AR−/− mice. The in vivo effect of BRL37344 on contractility of the nonfailing and failing heart in sheep was consistent with a beneficial effect of Na+-K+ pump stimulation in heart failure. Conclusions— The &bgr;3 AR mediates decreased &bgr;1 subunit glutathionylation and Na+-K+ pump stimulation in the heart. Upregulation of the receptor in heart failure may be a beneficial mechanism that facilitates the export of excess Na+.

Cardiomyocyte apoptosis is associated with increased wall stress in chronic failing left ventricle

AIMS We examined cardiomyocyte apoptosis in chronic heart failure (HF) and its possible link to elevated wall stress. METHODS AND RESULTS Moderate HF was produced in sheep by sequential coronary microembolization. Six months later, the animals remained in a stable compensated haemodynamic state of HF. Apoptosis of cardiomyocytes in left ventricles was verified using Western blotting based on increased expression of: the apoptosis-associated death receptor Fas (1.5-fold); its ligand (FasL, 2.0-fold); and an upstream protease caspase-8 (2.7-fold) as well as its active cleavage peptide, p20 (5.6-fold). Previously we have reported the elevated expression of caspase-3 in the same animal model. The occurrence of apoptotic cardiomyocytes (0.3%) was quantified by TUNEL assays. Haemodynamic analysis indicated that ventricular dilatation, without wall thickening, caused a 2-fold increase in LV wall stress which, together with LV end-diastolic pressure, was linearly correlated with expression of Fas/FasL. Immunohistochemical studies localized FasL and caspase-8 to intercalated discs, suggesting that wall stress may play a role in initiating cardiomyocyte apoptosis. CONCLUSION Apoptosis of cardiomyocytes in chronic HF is associated with increased wall stress, which may be responsible for the activation of a Fas/FasL and caspase-8 interaction in the region of intercalated discs.

Physiological Influences on Continuous Finger and Simultaneous Intra-arterial Blood Pressure

Because of the clinical and experimental utility of continuous finger blood pressure measurements and the need for accuracy, we tested the performance of a new hydraulic device in 22 consecutive hypertensive subjects during physiological and pharmacological interventions. Ipsilateral brachial intra-arterial pressure was monitored during rest, Valsalvas maneuver, static handgrip, and mental arithmetic and after sublingual glyceryl trinitrate. In excess of 40,000 blood pressure values were analyzed. Average bias (intra-arterial minus finger blood pressure) was 8.2 +/- 17.0 mm Hg (mean +/- SD, P = NS) for systolic and 2.8 +/- 10.4 mm Hg (P = NS) for diastolic pressure. Two-way ANOVA of biases with subject and task factors showed a subject effect (P < .001). Intersubject and intrasubject standard deviations of bias were 13.8 and 9.8 mm Hg systolic and 8.7 and 5.7 diastolic, respectively. Linear drift (millimeters of mercury per minute) of finger pressure was greater (P < .001) for systolic than diastolic pressure during static exercise and math and after glyceryl trinitrate. Coefficients of determination for blood pressure ranged from 0.4 +/- 0.3 to 0.8 +/- 0.3 during the tasks. We conclude that (1) noninvasive finger blood pressure faithfully follows intra-arterial changes but with clinically relevant offsets, (2) this technique is best suited for assessing pressure changes, (3) physiological and pharmacological interventions do not consistently affect finger pressure accuracy, (4) many reports of finger blood pressure measuring devices are based on direct readings obtained with inadequate system response characteristics, and (5) the tested instrument falls short of the standard requirements (bias < or = 5 +/- 8 mm Hg) for devices that measure intermittently.

Regional cardiac dysfunction is associated with specific alterations in inflammatory cytokines and matrix metalloproteinases after acute myocardial infarction in sheep.

Cardiac remodeling following myocardial infarction (MI) is a maladaptive process, fundamental to the progression of ischemic heart failure. The extent of remodeling is influenced by mechanical stress, inflammatory response and activation of matrix metalloproteinases (MMPs). This study examined regional association between these parameters in response to acute MI.Coronary ligation was performed in ten sheep. Sonomicrometer transducers measured segmental length in the infarcted, border and non-infarcted region. Regional tissue samples obtained 3 h post MI from six sheep were analysed using RT-PCR, gelatin zymography and Western blot. Six sham-operated sheep served as controls.Region-specific dilation and reduced contraction was associated with corresponding alterations in matrix molecules.IL-6 and MMP-9 mRNA were increased in the infarcted and border regions compared to controls.MMP-2 and TIMP-1 mRNA increased in non–infarcted myocardium and both correlated positively with segmental shortening. IL-6 mRNA levels, in contrast, were negatively associated with segmental shortening.In summary, inflammatory cytokines and MMPs are altered early after MI in a region-specific manner, and these changes correspond to acute regional myocardial dysfunction. Therapies for LV remodeling from the time of reperfusion may benefit from further understanding this portfolio of acute alterations.

Placebo-Controlled Biofeedback Blood Pressure Effect in Hypertensive Humans

The role of biofeedback in blood pressure control remains ill-defined because of nonspecific (placebo) effects, small study numbers, and the technical limitations of continuous pressure feedback. Clarification of its potential is awaited by those seeking a nonpharmacological approach to blood pressure control. This study examines the capability for systolic pressure lowering of 5 mm Hg or more using continuous pressure feedback in a statistical sample of untreated, well-characterized, mildly hypertensive individuals. Subjects were randomized in a double-blind study to active or placebo biofeedback. Placebo consisted of a modified contingency approach, using a partial disguise based on a digital high pass filter with 15 elements. Blood pressure-lowering capability was assessed during two laboratory sessions. Continuous visual feedback resulted in 11 of 28 subjects on active treatment and 12 of 28 on placebo treatment lowering their systolic pressure by 5 mm Hg or more (11 +/- 5.6 and 12 +/- 8.4 mm Hg, respectively; P = NS). Prestudy pressure was well-matched (153 +/- 9/97 +/- 4 and 154 +/- 8/98 +/- 4 mm Hg, respectively). An initial small difference in diurnal profile did not change. These findings indicate that among mildly hypertensive individuals, almost half can lower systolic pressure at will for short periods. This capability is independent of the real or placebo nature of the feedback signal. We conclude that there is no specific short-term biofeedback pressure-lowering capability in hypertensive individuals. Further exploration is needed to determine whether specific components of the placebo effect can be delineated, whether personality characteristics influence the response, and whether further biofeedback training can alter the outcome.

The effect of home training with direct blood pressure biofeedback of hypertensives: A placebo-controlled study

Background Home training in self-lowering of blood pressure using continuous blood pressure feedback has not previously been reported. Enhancement of laboratory-learned skills was hypothesized on the basis of outcomes from other intellectual, emotional and physical endeavours. Objective To examine the supplementary effect of home blood pressure biofeedback training. Design Thirty unmedicated, mild hypertensives participated in a randomized, double-blinded, modified contingency placebo-controlled study. Method After suitable screening and baseline blood pressure measurements subjects undertook eight laboratory biofeedback sessions and then 12 home training sessions over 4 weeks using continuous finger blood pressure monitoring. Results In the laboratory those being administered active therapy (n = 16) lowered systolic pressures by 5 ± 5.4 mmHg compared with a lowering of 4 ± 4.2 mmHg with placebo (NS). During the fourth week at home lowering for the active group (11 ± 8 mmHg) was greater than that with placebo (4 ± 6.2 mmHg, P = 0.017). Arm-cuff blood pressures were not statistically different for groups and with time but that of the active group was lower by 9 ± 15.4/7 ± 10.2 mmHg, which is a clinically relevant change, after home biofeedback. Conclusions The efficacy of self-lowering of systolic blood pressure in mild hypertensives by continuous feedback was enhanced by 6 mmHg with 4 weeks of practice at home. Standard arm-cuff blood pressure was reduced by a clinically relevant amount. The home environment proved cost effective for this ‘high-tech’ approach.

A stable ovine congestive heart failure model. A suitable substrate for left ventricular assist device assessment.

Similarities in coronary circulation and heart size of sheep to that of humans are specific advantages of a sheep model of congestive heart failure (CHF). CHF was created in 11 sheep (51 ± 4 kg) by selective sequential intracoronary injection of 90 μ microspheres under 1.5% isoflurane anesthesia. Hemodynamic characteristies were assessed at baseline, 4 weeks after establishment of CHF (ejection fraction [EF] < 35%, n = 11), and 26 weeks (n = 7) later. Baseline echocardiographic EF was 59 ± 5% and fell to 26 ± 5% after 5 ± 2 embolizations. The left ventricular (LV) pressure-volume relationship showed stable decreases in LV end-systolic elastance (Ees) and preload recruitable stroke work. Intravenous infusion of dobutamine increased Ees from 2.8 ± 1.7 to 4.3 ± 2.2 and 4.5 ± 1.4 mmHg/ml at heart rates of 140 and 160/min, respectively, at baseline. Increases of Ees (from 1.3. ± 0.5 to 2.3 ± 0.7 and 1.9 ± 0.5 mmHg/ml at heart rates of 140 and 160/min, respectively) with dobutamine under CHF conditions did not exceed Ees values at baseline without dobutamine. This response to dobutamine infusion did not change 26 weeks after establishment of CHF. This stable ovine CHF model is proposed for studies on the long-term effects of cardiac assist devices. ASAIO Journal 1997; 43:M408-M413.

Plasma Volume Contraction: A Significant Factor in Both Pregnancy-Associated Hypertension (Preeclampsia) and Chronic Hypertension in Pregnancy

The role of plasma volume in hypertension in pregnancy (pre-eclampsia) was investigated. Significant volume expansion from non-pregnant levels (16.5 +/- 1.60 ml/cm height) was present throughout pregnancy in 189 normal women, reaching 23.1 +/- 1.21 ml/cm at 33-36 weeks amenorrhoea. In another 40 initially normotensive pregnant women who developed hypertension, similar early volume expansion was followed by significant volume contraction in the third trimester, before evaluation of blood pressure in 29 (20.6 +/- 1.26 ml/cm), after it in 11 (18.6 +/- 1.27 ml/cm). Equivalent volume contraction was present in another 44 women studied only after hypertension developed in the third trimester. Oedema had no value as a clinical sign. In another 30 women with chronic hypertension, blood pressure was inversely related to plasma volume (r = 0.822) and to fetal growth (r = -0.710), which was directly related to plasma volume (r = 0.701). Plasma volume depletion plays a significant role in hypertension in pregnancy.

Microvascular Obstruction by Intracoronary Delivery of Mesenchymal Stem Cells and Quantification of Resulting Myocardial Infarction by Cardiac Magnetic Resonance

Intracoronary injection of mesenchymal stem cells (MSCs) has been proposed as a potential therapeutic option in repair of the ischemic- or infarct-damaged heart. However, because MSCs are large (rounded up cells are ≈22 to 25 μm diameter), an important consideration of this approach is the potential of these cells to induce myocardial damage by microvascular obstruction as has been reported previously.1 We present cardiac magnetic resonance (CMR) images, with corresponding biochemical and histological data from 2 healthy sheep in which 25 or 75×106 MSCs were injected through an over-the-wire balloon catheter into the mid left anterior descending coronary artery with the balloon inflated briefly before and during the infusion of the cell suspension. The MSC had been prepared as described previously and filtered with a 20-μm filter.2 Serial troponin levels were measured for 24 hours and, weight-adjusted gadolinium (Multihance; 0.1 mmol/kg) was injected 45 minutes before …