Influence of segmented vessel size due to limited imaging resolution on coronary hyperemic flow prediction from arterial crown volume.

Computational predictions of the functional stenosis severity from coronary imaging data use an allometric scaling law to derive hyperemic blood flow (Q) from coronary arterial volume (V), Q = αV(ß) Reliable estimates of α and ß are essential for meaningful flow estimations. We hypothesize that the relation between Q and V depends on imaging resolution. In five canine hearts, fluorescent microspheres were injected into the left anterior descending coronary artery during maximal hyperemia. The coronary arteries of the excised heart were filled with fluorescent cast material, frozen, and processed with an imaging cryomicrotome to yield a three-dimensional representation of the coronary arterial network. The effect of limited image resolution was simulated by assessing scaling law parameters from the virtual arterial network at 11 truncation levels ranging from 50 to 1,000 µm segment radius. Mapped microsphere locations were used to derive the corresponding relative Q using a reference truncation level of 200 µm. The scaling law factor α did not change with truncation level, despite considerable intersubject variability. In contrast, the scaling law exponent ß decreased from 0.79 to 0.55 with increasing truncation radius and was significantly lower for truncation radii above 500 µm vs. 50 µm (P< 0.05). Hyperemic Q was underestimated for vessel truncation above the reference level. In conclusion, flow-crown volume relations confirmed overall power law behavior; however, this relation depends on the terminal vessel radius that can be visualized. The scaling law exponent ß should therefore be adapted to the resolution of the imaging modality.

Perfusion territories subtended by penetrating coronary arteries increase in size and decrease in number toward the subendocardium.

Blood flow distribution within the myocardium and the location and extent of areas at risk in case of coronary artery disease are dependent on the distribution and morphology of intramural vascular crowns. Knowledge of the intramural vasculature is essential in novel multiscale and multiphysics modeling of the heart. For this study, eight canine hearts were analyzed with an imaging cryomicrotome, developed to acquire high-resolution spatial data on three-dimensional vascular structures. The obtained vasculature was skeletonized, and for each penetrating artery starting from the epicardium, the dependent vascular crown was defined. Three-dimensional Voronoi tessellation was applied with the end points of the terminal segments as center points. The centroid of end points in each branch allowed classification of the corresponding perfusion territories in subendocardial, midmyocardial, and subepicardial. Subendocardial regions have relatively few territories of about 0.5 ml in volume having their own penetrating artery at the epicardium, whereas the subepicardium is perfused by a multitude of small perfusion territories, in the order of 0.01 ml. Vascular volume density of small arteries up till 400 µm was 3.2% at the subendocardium territories but only 0.8% in the subepicardium territories. Their higher volume density corresponds to compensation for flow impeding forces by cardiac contraction. These density differences result in different scaling law properties of vascular volume and tissue mass per territory type. This novel three-dimensional quantitative analysis may form the basis for patient-specific computational models on coronary perfusion and aid the interpretation of image-based clinical methods for assessing the transmural perfusion distribution.

Degradation of the endothelial glycocalyx is associated with chylomicron leakage in mouse cremaster muscle microcirculation.

A thick endothelial glycocalyx contributes to the barrier function of vascular endothelium in macro- and microcirculation. We hypothesised in the current study that diet-induced hyperlipidaemia perturbs the glycocalyx, resulting in decreased dimensions of this layer and increased transendothelial lipoprotein leakage in capillaries. Glycocalyx thickness was measured in mouse cremaster muscle capillaries by intravital microscopy from the distance between flowing red blood cells and the endothelial surface. In control C57BL/6 mice on standard chow, glycocalyx thickness measured 0.58 ± 0.01 (mean ± SEM) µm, and no lipoproteins were observed in the tissue. After three months administration of an either mild or severe high-fat / high-cholesterol diet (HFC) to C57BL/6 and ApoE3-Leiden mice, circulating large lipoproteins appeared into the subendothelial space in an increasing proportion of cremaster capillaries, and these capillaries displayed reduced glycocalyx dimensions of 0.40 ± 0.02 and 0.30 ± 0.01 µm (C57BL/6 mice), and 0.37 ± 0.01 and 0.28 ± 0.01 µm (ApoE3-Leiden mice), after the mild and severe HFC diet, respectively. The chylomicron nature of the accumulated lipoproteins was confirmed by observations of subendothelial deposition of DiI-labeled chylomicrons in capillaries after inducing acute glycocalyx degradation by heparitinase in normolipidaemic C57BL/6 mice. It is concluded that while under control conditions the endothelial glycocalyx contributes to the vascular barrier against transvascular lipoprotein leakage in the microcirculation, diet-induced hyperlipidaemia reduces the thickness of the glycocalyx, thereby facilitating leakage of chylomicrons across the capillary wall.

Arrogance and ignorance in assessing scientific quality in a multidisciplinary academic medical centre.

Several academic institutions in the Netherlands and elsewhere develop indices to rank their scientists which will impact evaluation and steering of research. An important part of these indices is based on bibliometric indices. The development of such ranking indices is often seen as the prerogative of management and is kept out of the process where scientific instruments should be presented and evaluated: peer-reviewed journals. In this case the index of the author's institution is criticised both for the evasion of discussion as for the lack of compensation for bias related to discipline, gender and personal history. Furthermore, it is argued that the ranking based on 'numbers' rather than scientific contributions is detrimental to the motivation of the staff suffering under the several modi of bias, is counterproductive for interdisciplinary achievements and discourages young researchers in less scoring disciplines to find their way in the medical academic arena. (Neth Heart J 2010;18:319-22.).

Quantitative detection of cartilage surfaces and ligament geometry of the wrist using an imaging cryomicrotome system.

Biomechanical models may aid in improving diagnosis and treatment of wrist joint disorders. As input, geometrical information is required for model development. Previous studies acquired some elements of the average wrist joint geometry. However, there is a close geometric functional match between articulating surfaces and ligament geometry. Therefore, biomechanical models need to be fed with the geometric data of individual joints. This study is aimed at acquiring geometric data of cartilage surfaces and ligaments from individual wrist joints by using a cryomicrotome imaging system and the evaluation of inter- and intra-observer variability of the data. The 3D geometry of 30 cartilage surfaces and 15 ligaments in three cadaver wrists was manually detected and quantitatively reconstructed. The inter- and intra-observer variability of the cartilage surface detection was 0.14 and 0.19 mm, respectively. For the position of the radius attachment of the dorsal radiocarpal ligament (DRC), the observer variations were 0.12 and 0.65 mm, for intra-/inter-observer, respectively. For the DRC attachment on the triquetrum, the observer variations were 0.22 and 1.19 mm. Anatomic reconstruction from 3D cryomicrotome images offer a method to obtain unique geometry data of the entire wrist joint for modeling purposes.

A novel 3D multi-scale lineness filter for vessel detection.

The branching pattern and geometry of coronary microvessels are of high interest to understand and model the blood flow distribution and the processes of contrast invasion, ischemic changes and repair in the heart in detail. Analysis is performed on high resolution, 3D volumes of the arterial microvasculature of entire goat hearts, which are acquired with an imaging cryomicrotome. Multi-scale vessel detection is an important step required for a detailed quantitative analysis of the coronary microvasculature. Based on visual inspection, the derived lineness filter shows promising results on real data and digital phantoms, on the way towards accurate computerized reconstructions of entire coronary trees. The novel lineness filter exploits the local first and second order multi-scale derivatives in order to give an intensity-independent response to line centers and to suppress unwanted responses to steep edges.

Relation between branching patterns and perfusion in stochastic generated coronary arterial trees.

Biological variation in branching patterns is likely to affect perfusion of tissue. To assess the fundamental consequences of branching characteristics, 50 stochastic asymmetrical coronary trees and one non-stochastic symmetrical branching tree were generated. In the stochastic trees, area growth, A, at branching points was varied: A = random; 1.00; 1.10; 1.13 and 1.15 and symmetry, S, was varied: S = random; 1.00; 0.70; 0.58; 0.50 and 0.48. With random S and A values, a large variation in flow and volume was found, linearly related to the number of vessels in the trees. Large A values resulted in high number of vessels and high flow and volume values, indicating vessels connected in parallel. Lowering symmetry values increased the number of vessels, however, without changing flow, indicating a dominant connection of vessels in series. Both large A and small S values gave more realistic gradual pressure drops compared to the symmetrical non-stochastic branching tree. This study showed large variations in tree realizations, which may reflect real biological variations in tree anatomies. Furthermore, perfusion of tissue clearly depends on the branching rules applied.

Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalisation of myocardial perfusion areas.

A technique is presented for the 3D visualisation of the coronary arterial tree using an imaging cryomicrotome. After the coronary circulation of the excised heart was filled with a fluorescent plastic, the heart was frozen and mounted in the cryomicrotome. The heart was then sliced serially, with a slice thickness of 40 microm, and digital images were taken from each cutting plane of the remaining bulk material using appropriate excitation and emission filters. Using maximum intensity projections over a series of images in the cutting plane and perpendicular plane, the structural organisation of intramural vessels was visualised in the present study. The branching end in the smallest visible vessels, which define tissue areas that are well delineated from each other by 1-2 mm wide bands populated only by vessels less than 40 microm in diameter. The technique presented here allows further quantification in the future of the 3D structure of the coronary arterial tree by image analysis techniques.

Differential structural adaptation to haemodynamics along single rat cremaster arterioles.

We tested the hypothesis that under physiological conditions, arterioles match their diameter to the level of shear stress. Haemodynamic and anatomical data were obtained in segments of the first-order arteriole of the rat cremaster muscle. Along this segment of ~10 mm in length, local blood pressure decreased from 68 +/- 4 mmHg upstream to 54 +/- 3 mmHg downstream (n = 5). Pulse pressure decreased from 8.2 +/- 1.3 mmHg upstream to 4.1 +/- 0.6 mmHg downstream. At the same locations, an increase in arteriolar diameter was measured in vivo, from 179 +/- 4 microm upstream to 203 +/- 4 microm downstream (n = 10). In vitro pressure-diameter relations of maximally dilated vessels showed that the passive diameter was larger in downstream than upstream segments over a 15-125 mmHg pressure range (n = 18). The wall stress was similar for the upstream vs. downstream location: 266 +/- 16 vs. 260 +/- 14 mN mm-2. However, shear stress decreased from 30 +/- 5 to 21 +/- 5 dyn cm-2 (3.0 +/- 0.5 to 2.1 +/- 0.5 N m-2; n = 4) along the artery. In conclusion, these results demonstrate that shear stress is not the only factor in determining vascular calibre. We suggest that arteriolar calibre may rather depend on an interplay between shear stress and the local pressure profile.

Evidence for stretch-induced resistance increase of proximal coronary microcirculation.

We investigated the influence of stretch on regional hemodynamic parameters of the septal circulation. We used a similar experimental setup and mathematical model, as described previously (14). Five ventricular septa were isolated from anesthetized dogs, sutured to a biaxial stretching apparatus, and perfused with an oxygenated perfluorochemical emulsion at maximal vasodilation. Under unloaded and biaxially stretched conditions, flow and septal thickness (to index vascular volume) were measured continuously. Pressure was varied sinusoidally at 30, 50, and 70 mmHg with amplitude of 7.5 mmHg over frequencies ranging between 0.015 and 7 Hz. Admittance (flow/pressure) and capacitance (thickness/pressure) transfer functions were calculated and interpreted in terms of a two-compartmental model with volume-dependent resistances. Parameter estimation showed that the proximal resistance and compliance were unaffected, whereas the resistance of the proximal part of the microcirculation, including the small arterioles, increased with stretch. The effect of stretch on the distal resistance and capacitance, however, could not be determined unequivocally.

Vasomotor effects of arg-gly-asp (RGD) peptides are limited and not related to endothelium-derived hyperpolarizing factor-mediated relaxation in rat mesenteric arteries.

1. In the present study we tested the effect of arg-gly-asp (RGD) peptides on vasomotor responses in rat isolated mesenteric arteries. More specifically, the hypothesis was tested that RGD interaction with integrins mediates relaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). 2. The presence of the beta3 integrin subunit was shown by western blot analysis. To study its functional role, arteries (355 +/- 11 microm; n = 50) were mounted in a wire myograph set-up to measure isometric force generation. After blockade of nitric oxide synthesis with N(G)-nitro-L-arginine (0.1 mmol/L) and prostaglandin synthesis with indomethacin (10 micromol/L), methacholine (10 micromol/L) induced a transient relaxation within 1 min of 72 +/- 4.0% (as percentage of precontraction with phenylephrine; n = 27). 3. These responses were inhibited by a 60 mmol/L potassium buffer (18 +/- 6.0%; n = 6) or endothelium denudation (12 +/- 3.2%; n = 7), consistent with EDHF. 4. A function-blocking monoclonal antibody against the integrin beta3 chain did not affect relaxation. 5. The RGD peptides gly-arg-gly-asp-thr-pro (GRGDTP), gly-arg-gly-asp-ser (GRGDS) and cyclic RGD, ligands for the RGD binding site of integrins, also did not affect relaxation induced by methacholine. 6. Cyclic RGD increased contraction from 91 +/- 3 to 98 +/- 3% (as percentage of 120 mmol/L potassium). 7. In conclusion, these data show that vasomotor responses related to integrins are small and not involved in hyperpolarization attributed to EDHF in rat mesenteric artery.

Role of Rho-associated protein kinase in tone and calcium sensitivity of cannulated rat mesenteric small arteries.

The regulation of vascular tone includes modulation of contractile element calcium sensitivity. We tested the involvement of the Rho-associated protein kinase p160ROCK in tone and calcium sensitivity of cannulated rat mesenteric small arteries. These vessels developed basal tone and showed myogenic responses upon pressure steps, resulting from an increase in calcium in combination with a high contractile element calcium sensitivity. Y-27632, believed to be a specific p160ROCK inhibitor, caused concentration-dependent inhibition of basal tone, with near full inhibition at 3 microM. At this concentration, myogenic responses were absent and stepwise pressure elevation resulted in severe vascular distension. Y-27632 did not affect pressure-induced changes in intracellular calcium but rather reduced pressure-induced as well as phenylephrine-induced calcium sensitisation. Thus in the presence of the blocker, for a given calcium concentration, tone was greatly reduced, and the divergence in sensitivity between pressure and phenylephrine as stimuli on the one hand and potassium on the other disappeared. K+ (125 mM) and ionomycin still caused contraction in the presence of the p160ROCK blocker. These data show that in pressurised small arteries the Rho-p160ROCK pathway is active in the absence of vasoconstrictors, keeping the vessels in a state of high calcium sensitivity and basal tone.

Role of protein kinase C in myogenic calcium-contraction coupling of rat cannulated mesenteric small arteries.

1. The present study was designed to determine the role of protein kinase C (PKC) in the myogenic response of small arteries. In particular, we tested whether inhibition of PKC reverses the previously found pressure-induced elevation of contractile element calcium sensitivity. 2. Rat mesenteric small arteries were cannulated and pressurized. The internal diameter was continuously monitored with a video camera and intracellular calcium levels were measured by means of fura-2. Myogenic responses were observed when the pressure was raised stepwise from 20 to 60 and then to 100 mmHg in physiological saline solution and during application of phenylephrine (0.1 or 1 micromol/L) or potassium (36 mmol/L). 3. The PKC inhibitors H-7 (20 micromol/L), staurosporine (100 nmol/L) and calphostin C (10 nmol/L) all completely abolished the myogenic response. Whereas staurosporine caused an ongoing reduction in intracellular calcium, pressure-induced calcium transients were not affected by either H-7 or calphostin C. In particular, the slope of the wall tension-calcium relationship remained similar in the presence of both H-7 and calphostin C, despite an upward shift of this relationship to higher calcium levels in the case of calphostin C. 4. These results show that activity of PKC isoform(s) is essential for myogenic calcium-contraction coupling.

Impact of current cryopreservation procedures on mechanical and functional properties of human aortic homografts.

We evaluated the impact f standard cryopreservation on mechanical and functional properties of human aortic homografts. From 14 human heart-valve donors, the thoracic descending aorta was obtained. Effects of cryopreservation on mechanical (elastic properties and breaking stress) and smooth muscle cell (SMC) and endothelium function were tested. Cryopreservation (cryo) did not significantly affect Young's modulus of elastin (fresh: 3.1 +/- 1.0, cryo: 2.7 +/- 0.9 x 10(5) Nm(-2)), collagen recruitment pressure (fresh: 1.1 +/- 0.3, cryo: 1.1 +/- 0.4 x 10(4) Nm(-2)), distensibility (fresh: 3.8 +/- 1.8, cryo: 3.6 +/- 1.6 x 10(5) N(-1)m2), or breaking stress (fresh: 2.4 +/- 1.0, cryo: 2.2 +/- 1.0 x 10(6) Nm(-2)). Following explantation, no endothelium-dependent relaxation was found. SMC function and endothelium-independent relaxation were mainly intact after explantation but significantly decreased after cryopreservation. Aortic mechanical properties are not influenced by cryopreservation. Following explantation, almost no endothelial cell function is present, and SMC contractility is strongly affected after cryopreservation.

Laser pulse impact on rat mesenteric blood vessels in relation to laser treatment of port wine stain.

BACKGROUND AND OBJECTIVE: To study the impact of laser pulses on animal microvasculature as a model for laser treatment of port wine stains. STUDY DESIGN/MATERIALS AND METHODS: Rat mesenteric blood vessels were irradiated with a laser pulse (585 nm, 0.2-0.6 ms pulse duration, 0.5-30 J/cm(2) radiant exposure). Video microscopy was used to assess vessel dilation, formation of intravascular thrombi, bubble formation, and vessel rupture. Changes in reflection during a laser pulse were measured by simultaneously recording the temporal behavior of the incident and reflected signals. RESULTS: A threshold radiant exposure of approximately 3 J/cm(2) was found for changes in optical properties of blood in vivo, confirming previous in vitro results. Often, laser exposure induced a significant increase in vessel diameter, up to three times the initial diameter for venules and four times for arterioles, within 200 ms after laser exposure. Arterioles were more likely to dilate than venules. Sometimes, immediately after the pulse, round structures, interpreted as being gas bubbles, were seen within the vessel lumen. CONCLUSIONS: A variety of phenomena can occur when blood vessels of sizes comparable to those in port wine stains are irradiated with laser pulses as used in port wine stain treatment. Thrombus formation and vessel rupture have been described before from histological sections of laser-irradiated port wine stains. However, vessel dilation and formation of non-transient gas bubbles as found in this study have not been described before.

Biophysical properties of the normal-sized aorta in patients with Marfan syndrome: evaluation with MR flow mapping.

PURPOSE: To investigate the feasibility of magnetic resonance (MR) flow mapping in the assessment of aortic biophysical properties in patients with Marfan syndrome and to detect differences in biophysical properties in the normal-sized aorta distal to the aortic root between these patients and matched control subjects. MATERIALS AND METHODS: Seventy-eight patients with Marfan syndrome with aortic root dilatation and 23 matched control subjects underwent MR flow mapping in four locations in the normal-sized aorta (1, ascending aorta; 2, thoracic descending aorta; 3, descending aorta at the level of the diaphragm; and 4, abdominal descending aorta). Distensibility at each location and flow wave velocity between locations were calculated. RESULTS: Compared with the control subjects, patients with Marfan syndrome had decreased aortic distensibility at three of the four locations (levels 1, 2, and 4; P <.05) and increased flow wave velocity between all locations (P <.05) in the aorta. In patients with Marfan syndrome, flow wave velocity was also significantly increased along the entire aortic tract beyond the aortic root (from level 1 to level 4). CONCLUSION: MR imaging reveals abnormal biophysical properties of the normal-sized aorta in patients with Marfan syndrome. Monitoring of these properties is relevant for evaluating disease progression and treatment options.

Stenosis differentially affects subendocardial and subepicardial arterioles in vivo.

The presence of a coronary stenosis results primarily in subendocardial ischemia. Apart from the decrease in coronary perfusion pressure, a stenosis also decreases coronary flow pulsations. Applying a coronary perfusion system, we compared the autoregulatory response of subendocardial (n = 10) and subepicardial (n = 12) arterioles (<120 microm) after stepwise decreases in coronary arterial pressure from 100 to 70, 50, and 30 mmHg in vivo in dogs (n = 9). Pressure steps were performed with and without stenosis on the perfusion line. Maximal arteriolar diameter during the cardiac cycle was determined and normalized to its value at 100 mmHg. The initial decrease in diameter during reductions in pressure was significantly larger at the subendocardium. Diameters of subendocardial and subepicardial arterioles were similar 10--15 s after the decrease in pressure without stenosis. However, stenosis decreased the dilatory response of the subendocardial arterioles significantly. This decreased dilatory response was also evidenced by a lower coronary inflow at similar average pressure in the presence of a stenosis. Inhibition of nitric oxide production with N(G)-monomethyl-L-arginine abrogated the effect of the stenosis on flow. We conclude that the decrease in pressure caused by a stenosis in vivo results in a larger decrease in diameter of the subendocardial arterioles than in the subepicardial arterioles, and furthermore stenosis selectively decreases the dilatory response of subendocardial arterioles. These two findings expand our understanding of subendocardial vulnerability to ischemia.

Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL.

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500-1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL (n = 4), moderate oxLDL (6-h oxidation with CuSO(4), n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (SOD) and catalase (n = 8). Capillary tube hematocrit increased from 0.16 +/- 0.03 to 0.37 +/- 0.05 and from 0.15 +/- 0.01 to 0.31 +/- 0.03 after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 +/- 1.9 to 13.8 +/- 3 and from 10.7 +/- 2.1 to 16.3 +/- 3.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with SOD and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.

Role of variability in microvascular resistance on fractional flow reserve and coronary blood flow velocity reserve in intermediate coronary lesions.

BACKGROUND: Fractional flow reserve (FFR) and coronary blood flow velocity reserve (CFR) represent physiological quantities used to evaluate coronary lesion severity and to make clinical decisions. A comparison between the outcomes of both diagnostic techniques has not been performed in a large cohort of patients with intermediate coronary lesions. METHODS AND RESULTS: FFR and CFR were assessed in 126 consecutive patients with 150 intermediate coronary lesions (between 40% and 70% diameter stenosis by visual assessment). Agreement between outcomes of FFR and CFR, categorized at cut-off values of 0.75 and 2.0, respectively, was observed in 109 coronary lesions (73%), whereas discordant outcomes were present in 41 lesions (27%). In 26 of these 41 lesions, FFR was <0.75 and CFR>or=2.0 (group A); in the remaining 15 lesions, FFR was >or=0.75 and CFR<2.0 (group B). Minimum microvascular resistance, defined as the ratio of mean distal pressure to average peak blood flow velocity during maximum hyperemia, showed a large variability (overall range, 0.65 to 4.64 mm Hg x cm(-1) x s(-1)) and was significantly higher in group B than in group A (2.42+/-0.77 versus 1.91+/-0.70 mm Hg x cm(-1) x s(-1); P:=0.034). CONCLUSIONS: Our findings demonstrate the prominent role of microvascular resistance in modulating the relationship between FFR and CFR and emphasize the importance of combined pressure and flow velocity measurements to evaluate coronary lesion severity and microvascular involvement.