Effects of vitamin D2 or D3 supplementation on glycaemic control and cardiometabolic risk among people at risk of type 2 diabetes: results of a randomized double-blind placebo-controlled trial.

AIMS: To investigate the effect of short-term vitamin D supplementation on cardiometabolic outcomes among individuals with an elevated risk of diabetes. METHODS: In a double-blind placebo-controlled randomized trial, 340 adults who had an elevated risk of type 2 diabetes (non-diabetic hyperglycaemia or positive diabetes risk score) were randomized to either placebo, 100,000 IU vitamin D2 (ergocalciferol) or 100,000 IU vitamin D3 (cholecalciferol), orally administered monthly for 4 months. The primary outcome was change in glycated haemoglobin (HbA1c) between baseline and 4 months, adjusted for baseline. Secondary outcomes included: blood pressure; lipid levels; apolipoprotein levels; C-reactive protein levels; pulse wave velocity (PWV); anthropometric measures; and safety of the supplementation. RESULTS: The mean [standard deviation (s.d.)] 25-hydroxyvitamin D [25(OH)D]2 concentration increased from 5.2 (4.1) to 53.9 (18.5) nmol/l in the D2 group, and the mean (s.d.) 25(OH)D3 concentration increased from 45.8 (22.6) to 83.8 (22.7) nmol/l in the D3 group. There was no effect of vitamin D supplementation on HbA1c: D2 versus placebo: -0.05% [95% confidence interval (CI) -0.11, 0.02] or -0.51 mmol/mol (95% CI -1.16, 0.14; p = 0.13); D3 versus placebo: 0.02% (95% CI -0.04, 0.08) or 0.19 mmol/mol (95% CI -0.46, 0.83; p = 0.57). There were no clinically meaningful effects on secondary outcomes, except PWV [D2 versus placebo: -0.68 m/s (95% CI -1.31, -0.05); D3 versus placebo -0.73 m/s (95% CI -1.42, -0.03)]. No important safety issues were identified. CONCLUSIONS: Short-term supplementation with vitamin D2 or D3 had no effect on HbA1c. The modest reduction in PWV with both D2 and D3 relative to placebo suggests that vitamin D supplementation has a beneficial effect on arterial stiffness.

Characterisation of Elastic and Acoustic Properties of an Agar-Based Tissue Mimicking Material.

As a first step towards an acoustic localisation device for coronary stenosis to provide a non-invasive means of diagnosing arterial disease, measurements are reported for an agar-based tissue mimicking material (TMM) of the shear wave propagation velocity, attenuation and viscoelastic constants, together with one dimensional quasi-static elastic moduli and Poisson's ratio. Phase velocity and attenuation coefficients, determined by generating and detecting shear waves piezo-electrically in the range 300 Hz-2 kHz, were 3.2-7.5 ms(-1) and 320 dBm(-1). Quasi-static Young's modulus, shear modulus and Poisson's ratio, obtained by compressive or shear loading of cylindrical specimens were 150-160 kPa; 54-56 kPa and 0.37-0.44. The dynamic Young's and shear moduli, derived from fitting viscoelastic internal variables by an iterative statistical inverse solver to freely oscillating specimens were 230 and 33 kPa and the corresponding relaxation times, 0.046 and 0.036 s. The results were self-consistent, repeatable and provide baseline data required for the computational modelling of wave propagation in a phantom.

Carotid atherosclerotic plaque characterisation by measurement of ultrasound sound speed in vitro at high frequency, 20 MHz.

This study aimed to utilise a tissue mimicking material (TMM) in order to embed in vitro carotid plaque tissue so that its acoustic properties could be assessed. Here, an International Electrotechnical Commission (IEC) agar-based TMM was adapted to a clear gel by removal of the particulates. This clear TMM was measured with sound speed at 1540 ms(-1) and an attenuation coefficient of 0.15 dB cm(-1)MHz(-1). Composite sound speed was then measured through the embedded material using a scanning acoustic microscope (SAM). Both broadband reflection and transmission techniques were performed on each plaque specimen in order to ensure the consistency of the measurement of sound speed, both at 21 °C and 37 °C. The plaque was measured at two temperatures to investigate any effect on the lipid content of the plaque. The contour maps from its associated attenuation plots were used to match the speed data to the photographic mask of the plaque outline. This physical matching was then used to derive the sound speed from the percentage composition seen in the histological data by solution of simultaneous equations. Individual speed values for five plaque components were derived; TMM, elastin, fibrous/collagen, calcification and lipid. The results for derived sound speed in the TMM were consistently close to the expected value of soft tissue, 1540 ms(-1). The fibrous tissue showed a mean value of 1584 ms(-1) at 37 °C. The derived sound speeds for elastic and lipid exhibited large inter-quartile ranges. The calcification had higher sound speed than the other plaque components at 1760-2000 ms(-1). The limitations here lay in the difficulties in the matching process caused by the inhomogeneity of the plaque material and shrinkage during the histological process. Future work may concentrate on more homogeneous material in order to derive sound speed data for separate components. Nevertheless, this study increases the known data ranges of the individual components within a plaque. This information may be used help to assess the mechanical properties and structural integrity and its associated vulnerability or risk of embolization in future diagnostic ultrasound techniques.

Cardiac function in 10-year-old twins following different fetal therapies for twin-twin transfusion syndrome.

OBJECTIVES: To compare cardiac function at 10 years of age in four groups of monochorionic diamniotic (MCDA) twin pairs: uncomplicated MCDA twins (n = 6) (Group 1); twins that had had twin-twin transfusion syndrome (TTTS) managed by amnioreduction (TTTS-amnio, n = 9) (Group 2) or laser photocoagulation (TTTS-laser, n = 10) (Group 3); and dichorionic diamniotic controls (DCDA, n = 6) (Group 4). METHODS: Echocardiograms optimizing apical four-chamber and short-axis left ventricular views were stored for offline speckle-tracking analysis, blinded to twin type. Myocardial long-axis shortening and lengthening velocities were measured using pulsed Doppler ultrasound at the cardiac base. M-mode measurements of fractional shortening (short axis) and maximal excursion of the atrioventricular annulus (four-chamber) were recorded. Syngo Vector Velocity Imaging software tracked left ventricular myocardial motion offline to produce free wall strain, strain rate and rotation. Intertwin pair and group differences were investigated using ANOVA. RESULTS: Cardiac measurements were within the normal ranges for 10-year-olds. No significant within-twin-pair and intergroup differences were found in current size, heart rates, strain or strain rate. Compared to DCDA controls, TTTS twins showed less cardiac rotation (TTTS-laser, P < 0.001 and TTTS-amnio, P = 0.054) with significant intertwin reduction in the ex-recipient (TTTS-amnio, P = 0.006) and larger MCDA twins (P = 0.027) compared with their cotwins. A similar pattern was seen in left ventricular early diastolic mitral valve tissue velocity (MVE') in all monochorionic groups, but only achieving significance in TTTS-amnio twins (P = 0.037). Intrapair differences in rotation and MVE' were significantly different following treatment at Quintero stages III or IV. CONCLUSIONS: Within-twin-pair patterns of left ventricular rotation and diastolic function differ at 10 years of age in ex-recipients of TTTS twins treated with amnioreduction compared with those treated by laser photocoagulation and controls. .

Vascular programming in twins: the effects of chorionicity and fetal therapy for twin-to-twin transfusion syndrome.

We assessed vascular programming in genetically identical monochorionic twin pairs with twin-to-twin transfusion syndrome (TTTS) treated differently in utero by serial amnioreduction or fetal laser arterial photocoagulation. This case-control study re-assessed four twin groups at median 11 years comprising 20 pairs of monochorionic diamniotic twins: nine treated by amnioreduction (TTTS-amnio) and eleven by laser (TTTS-laser) with seven monochorionic and six dichorionic control pairs. Outcome measures were current blood pressure (BP), brachio-radial arterial stiffness derived from pulse wave velocity (PWV), resting microcirculation (Flux) and response to heating and post-occlusive reactive hyperaemia measured using laser Doppler. Potential confounders [PWV and BP at first study, current height, weight, heart rate and twin type (ex-recipient, ex-donor or heavier/lighter of pair)] were accounted for by Mixed Linear Models statistical methodology. PWV dichorionic > monochorionic (P = 0.024); systolic and diastolic BP dichorionic > TTTS-amnio and TTTS-laser (P = 0.004, P = 0.02 and P = 0.005, P = 0.02, respectively). Within-twin pair pattern of PWV discordance was similar in laser treated and dichorionic controls (heavier-born > lighter), opposite to TTTS-amnio and monochorionic controls. Flux monochorionic > dichorionic (P = 0.044) and heavier > lighter-born (P = 0.024). TTTS-laser and dichorionic diamniotic showed greatest hyperaemic responses (dichorionic > TTTS-amnio or monochorionic controls (P = 0.007, P = 0.025). Hyperaemic responses were slower in heavier-born twins (P = 0.005). In summary, monochorionic twins had lower BP, arterial stiffness and increased resting vasodilatation than dichorionic twins implying shared fetal circulation affects vascular development. Vascular responses in laser-TTTS were similar to dichorionic and opposite to TTTS-amnio suggesting a lasting effect of fetal therapy on vascular health.

Ageing of the conduit arteries.

Conduit arteries become stiffer with age due to alterations in their morphology and the composition of the their major structural proteins, elastin and collagen. The elastic lamellae undergo fragmentation and thinning, leading to ectasia and a gradual transfer of mechanical load to collagen, which is 100-1000 times stiffer than elastin. Possible causes of this fragmentation are mechanical (fatigue failure) or enzymatic (driven by matrix metallo proteinases (MMP) activity), both of which may have genetic or environmental origins (fetal programming). Furthermore, the remaining elastin itself becomes stiffer, owing to calcification and the formation of cross-links due to advanced glycation end-products (AGEs), a process that affects collagen even more strongly. These changes are accelerated in the presence of disease such as hypertension, diabetes and uraemia and may be exacerbated locally by atherosclerosis. Raised MMP activity, calcification and impaired endothelial function are also associated with a high level of plasma homocysteine, which itself increases with age. Impaired endothelial function leads to increased resting vascular smooth muscle tone and further increases in vascular stiffness and mean and/or pulse pressure. The effect of increased stiffness, whatever its underlying causes, is to reduce the reservoir/buffering function of the conduit arteries near the heart and to increase pulse wave velocity, both of which increase systolic and pulse pressure. These determine the peak load on the heart and the vascular system as a whole, the breakdown of which, like that of any machine, depends more on the maximum loads they must bear than on their average. Reversing or stabilising the increased arterial stiffness associated with age and disease by targeting any or all of its causes provides a number of promising new approaches to the treatment of systolic hypertension and its sequelae, the main causes of mortality and morbidity in the developed world.

Residual strains in conduit arteries.

Residual strains and stresses are those that exist in a body when all external loads are removed. Residual strains in arteries can be characterized by the opening angle of the sector-like cross-section which arises when an unloaded ring segment is radially cut. A review of experimental methods for measuring residual strains and the main results about the variation of the opening angle with arterial localization, age, smooth muscle activity, mechanical environment and certain vascular pathologies are presented and discussed. It is shown that, in addition to their well-established ability to homogenize the stress field in the arterial wall, residual strains make arteries more compliant and thereby improve their performance as elastic reservoirs and ensure more effective local control of the arterial lumen by smooth muscle cells. Finally, evidence that, in some cases, residual strains remain in arteries even after they have been cut radially is discussed.

A hypothesis about a mechanism for the programming of blood pressure and vascular disease in early life.

1. There is now a great deal of evidence that people whose weight at birth was low tend to have higher blood pressure and increased risk of death from cardiovascular disease as adults. 2. We argue that, in fetuses whose growth is impaired, synthesis of elastin in the walls of the aorta and large arteries is deficient and that this deficiency leads to permanent changes in the mechanical properties of these vessels. 3. Over a lifetime, such changes could predispose an individual to higher blood pressure and cardiovascular disease.

Assessing the homogeneity of the elastic properties and composition of the pig aortic media.

Most previous studies of arterial wall elasticity and rheology have assumed that the properties of the wall are uniform across the thickness of the media and, therefore, that the relationship between stress and strain may be described by a constitutive equation based on a single strain energy function. The few studies where this assumption has been questioned, focussed on differences between the adventitia and the media rather than on differences within the media itself. Here, we report in vitro elasticity and residual strain measurements performed separately on the inner and outer half of the pig aortic media, together with a histomorphometric assessment of the radial distribution of elastin, collagen and smooth muscle cell numbers. Although we found that the pressure-diameter relationships of the two halves were dissimilar, when allowance was made for their different unloaded dimensions, their material properties agreed closely, a result in keeping with the observed uniform radial distribution of scleroprotein and vascular smooth muscle. We also found a difference in the opening angle (which is often taken as a measure of residual strain) between the inner and outer medial halves. However, strain analysis showed that the opening angle is an extremely sensitive measure of residual strain and that the difference in the actual magnitudes of residual strain between the two halves of the media was small. We conclude that the media of the porcine thoracic aorta has similar elastic properties throughout its thickness and that this uniformity is matched by a uniform distribution of matrix protein and vascular smooth muscle cells. Furthermore, the distribution of strain in the media can adequately be described by a single-layer model with uniform elastic properties throughout its thickness.

Improving vascular grafts: the importance of mechanical and haemodynamic properties.

In the last 40 years, as techniques and materials have improved, the success rate of vascular prostheses with a diameter greater than 6mm has risen steadily, 5-year survival rates exceeding 95% in most centres. With smaller grafts no comparable improvement has occurred, the majority failing within 5 years, usually as a result of intimal hyperplasia and, ultimately atherosclerosis, in and around the downstream anastomosis. Clinical evidence suggests that the patency rates of small grafts are improved by matching the elastic properties of the graft to that of the artery into which it is placed. Although there is little reliable evidence that 'elastic mismatch' per se is the cause of intimal hyperplasia, it is generally accepted that mechanical factors are important in its genesis. These include disturbed flow at the anastomosis leading to fluctuations in shear stress at the endothelium (a known cause of intimal hyperplasia in normal arteries), injury due to suturing and stress concentration at the anastomosis. Few suitable materials or techniques have yet been developed to improve the long-term survival rates of small grafts. Recent advances in tissue engineering in which prostheses are manufactured by culturing vascular smooth muscle cells on a tubular scaffold of biodegradable polymer may ultimately make it possible to manufacture biologically and haemodynamically compatible grafts with diameters as small as 1mm.

Impaired synthesis of elastin in walls of aorta and large conduit arteries during early development as an initiating event in pathogenesis of systemic hypertension.

There is much evidence that people who had low birthweight tend to have higher blood pressure in later life. However, the mechanisms that mediate this relation are unknown. We argue that, in fetuses whose growth is impaired, synthesis of elastin in the walls of the aorta and large arteries may be deficient, and that this deficiency would lead to permanent changes in the mechanical properties of these vessels. Over a lifetime, such changes could predispose an individual to higher blood pressure, increased left-ventricular mass, and cardiovascular disease.

Comparison of different methods for the determination of the true wave propagation coefficient, in rubber tubes and the canine thoracic aorta.

The results from studies of wave propagation in large arteries carried out over the last 25 years have shown that there is a good agreement among values of the imaginary part of the complex propagation coefficient, as expressed by pressure or flow-rate wave propagation velocity. However, there is considerable disparity among estimations of the degree of wave attenuation, the real part of the propagation coefficient. In order to determine whether this disparity is due to differences inherent in the various methods used to measure true wave propagation coefficients or whether it is caused by differences in experimental conditions, we have compared three techniques for determining true pulse wave propagation coefficients the three-point method, the occlusion method and a recently described iterative procedure. In addition, the results were compared to apparent propagation coefficients calculated without accounting for reflections. Measurements were carried out using each method in turn on a rubber tube of known transmission characteristics in which the magnitude of reflections was small. The iterative procedure and the three-point method were also compared under conditions of strong reflection. In the tube, the values of propagation velocity and attenuation coefficient determined by each method were similar. Although some discrepancies were noted, they did not amount to a systematic trend. The iterative procedure and the occlusion method were also used to analyse measurements on the thoracic aorta of three anaesthetized greyhounds. In the animal experiments, in spite of increased scatter, partly due to the variation between dogs, the two methods for determining true pulse-wave propagation yielded similar results. Since the differences between our estimates of propagation coefficients obtained by the methods tested are small with respect to those found when comparing the results from several reports in the literature, we conclude that any discrepancies between studies cannot be due to problems associated with the methods themselves but must have been caused by variations in experimental conditions or by other unknown artefacts.

Experimental investigation of the distribution of residual strains in the artery wall.

Arterial wall stresses are thought to be a major determinant of vascular remodeling both during normal growth and throughout the development of occlusive vascular disease. A completely physiologic mechanical model of the arterial wall should account not only for its residual strains but also for its structural nonhomogeneity. It is known that each layer of the artery wall possesses different mechanical properties, but the distribution of residual strain among the different mechanical components, and thus the true zero stress state, remain unknown. In this study, two different sets of experiments were carried out in order to determine the distribution of residual strains in artery walls, and thus the true zero stress state. In the first, collagen and elastin were selectively eliminated by chemical methods and smooth muscle cells were destroyed by freezing. Dissolving elastin provoked a decrease in the opening angle, while dissolving collagen and destroying smooth muscle cells had no effect. In the second, different wall layers of bovine carotid arteries were removed from the exterior or luminal surfaces by lathing or drilling frozen specimens, and then allowing the frozen material to thaw before measuring residual strain. Lathing material away from the outer surface caused the opening angle of the remaining inner layers to increase. Drilling material from the inside caused the opening angle of the remaining outer layers to decrease. Mechanical nonhomogeneity, including the distribution of residual strains, should thus be considered as an important factor in determining the distribution of stress in the artery wall and the configuration of the true zero stress state.

The effect of age on residual strain in the rat aorta.

Residual stress is observed in many parts of the cardiovascular system and is thought to reduce transmural stress gradients due to intravascular pressure. Its development is closely associated with normal growth and pathological remodeling, although there appear to be few previous reports of the relationship between aging and residual stress. We have estimated residual strain (an indicator of the magnitude of residual stress) at ten sites along the aorta of rats aged 2.5 to 56 weeks by measuring the degree to which rings of vessel spring open when cut (opening angle). At all ages the opening angle decreased along the aorta, reaching a minimum near the renal arteries and increasing toward the aorto-iliac bifurcation, a result that confirms previous studies. During growth, although the unloaded circumference of the aorta increased steadily, the wall thickness and medial surface area fell to a minimum at the age of 6 weeks before continuing a steady increase. Similarly, the opening angle decreased between the ages of 2.5 and 6 weeks, thereafter increasing with age. In the abdominal aorta, a strong correlation between opening angle and wall thickness relative to midwall radius (h/R) was seen; whereas in the thoracic segment, in which no increase in h/R with age occurred, no such relationship was found. These observations are in keeping with a recently proposed hypothesis that residual stress will change in response to growth-related changes in vessel geometry driven by a tendency to minimize the nonuniform stress distribution inevitably found in pressurized thick-walled cylinders.

Analysis of the strain and stress distribution in the wall of the developing and mature rat aorta.

The variation of wall stress distribution with age in the thoracic and abdominal aortas of normotensive rats was studied. Dimensions of the zero-stress configurations were measured at the ages of 4, 8, 12, 20, and 52 weeks. Using data from previously published inflation tests, the circumferential stress-strain relationship was obtained in each age group. The calculated stress distribution showed that the average circumferential stress remained practically constant after the age of 20 weeks. The circumferential stress at the innermost part of the arterial wall was greater than the stress at the outermost part, but the difference was maintained at a moderate level with adjustments in the zero-stress configuration. It is speculated that, after the age of 20 weeks, changes in arterial geometry and rheological properties tend to maintain a constant stress distribution under varying conditions of loading. This distribution was achieved by enhanced growth at the inner part of the media in comparison with the growth at its outer margins and suggests that during development and maturity, the growth of the aorta is modulated by circumferential stress.

Desmin expression in rhabdomyosarcoma: influence of the desmin clone and immunohistochemical method.

AIM: To determine which, if any, of five commercially available desmin clones is most reliable at labelling desmin filaments and whether the enhanced polymer one step (EPOS) method of labelling is of any advantage in the routine diagnostic laboratory. METHODS: Thirty four rhabdomyosarcomas from the files at The Hospital for Sick Children, Great Ormond Street, London, were studied. Four different desmin clones, DE-R-11, D33, DE-U-10, and PDE, were applied to each using the conventional extravidin biotin peroxidase method. The D33 clone was also applied using the EPOS method. RESULTS: The EPOS method incorporating D33 persistently scored more cells as desmin positive and was positive in four cases which were negative on staining with the other clones. CONCLUSIONS: The D33 desmin clone used with the EPOS method is more reliable for identifying desmin filaments in tumours than other desmin antibodies tested. Different desmin clones using a routine technique label different rhabdomyosarcoma cells and therefore it is justifiable to use more than one clone.

The relationship between wall tension, lamellar thickness, and intercellular junctions in the fetal and adult aorta: its relevance to the pathology of dissecting aneurysm.

It is known that the distribution of stress and strain in the vessel wall is not uniform. We believe that this explains the location of the plane of dissection in dissecting aneurysms of large elastic arteries. We have investigated the effects of non-uniformity of stress and strain on the thickness of each elastic lamella and on the distribution of intercellular junctions in the media of developing and adult rats, to seek evidence to support this hypothesis. Intercellular junctions were identified by transmission electron microscopy of whole wall sections. A morphometric study of elastic tissue distribution was made on an image analysis computer. Differences were analysed using one-way analysis of variance. There are between six and eight elastic lamellae in the aorta of rats. In the fetus, only the internal elastic lamella is complete; the others were not fully formed by term. In the adult, the inner five elastic lamellae were thicker than the remaining two or three, and smooth muscle cells in the thicker lamellar units had more cell-cell contacts of all types examined. These data support the concept of a difference in stress-resisting properties of the aortic wall on the junctions between the inner two-thirds and the outer third of the media. The findings indicate that, as proposed in theoretical models the innermost lamellae support the high tension. In the adult aorta, the structure is modified to enhance the capacity to resist stress in the internal two-thirds of the media.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of an in-vitro perfusion system for studies on cultured cells.

The design and use of a perfusion system, using a modified flow chamber for studies on cultured animal cells, is described. Rat thoracic aorta smooth muscle cells were isolated by an explant method and grown on Thermanox coverslips. These were introduced into the flow chamber. A flow rate of 25ml/min and a shear stress of 14.6 dynes/cm2 (12 dyne = 10 microN) (both within physiological limits) were maintained. Cells remained attached to the coverslips after 8h of perfusion with culture medium. The effect of exposing rat smooth muscle cells to the cardiovascular toxin, allylamine, is also described. The components of the system are routinely available, simple to clean, easy to assemble and sterilize. The incorporation of an in-line sensor that monitors pH, PO2, PCO2 and temperature ensures that the perfusion conditions remain within physiological limits. Automation means that minimal supervision is required. This system provides a potential mechanism in which cultured vascular cells may be perfused under controlled haemodynamic conditions, and their response to a cytotoxin may be evaluated.