Erik L. Ritman

Enhanced coronary vasa vasorum neovascularization in experimental hypercholesterolemia.

Coronary arteries contain a network of vasa vasorum in the adventitia. The three-dimensional anatomy of the vasa vasorum in early coronary atherosclerosis is unknown. This study was designed to visualize and quantitate the three-dimensional spatial pattern of vasa vasorum in normal and experimental hypercholesterolemic porcine coronary arteries, using a novel computed tomography technique. Animals were killed after being fed either a high cholesterol diet (n = 4) or a control diet (n = 4) for 12 wk. The proximal left anterior descending coronary artery was removed from the heart, scanned, and reconstructed, and quantitation of vasa vasorum density was performed. Two different types of vasa vasorum were defined: first-order vasa vasorum ran longitudinally parallel to the vessel and second-order originated from first-order vasa circumferentially around the vessel wall. Compared with controls in hypercholesterolemic coronary arteries, there was a significant increase in the area of the vessel wall (3.86+/-0.22 vs. 8.07+/-0.45 mm2, respectively, P < 0.01) and in the density of vasa vasorum (1. 84+/-0.05/mm2 vs. 4.73+/-0.24/mm2; respectively, P = 0.0001). This occurred especially by an increase of second-order vasa vasorum and disorientation of normal vasa vasorum spatial pattern. This study suggests that adventitial neovascularization of vasa vasorum occurs in experimental hypercholesterolemic coronary arteries and may be a part of the early atherosclerotic remodeling process.

Three-dimensional imaging of vasculature and parenchyma in intact rodent organs with X-ray micro-CT

A microcomputed tomography (micro-CT) scanner, which generates three-dimensional (3-D) images consisting of up to a billion cubic voxels, each 5-25 micron on a side, and which has isotropic spatial resolution, is described. Its main components are a spectroscopic X-ray source that produces selectable primary emission peaks at approximately 9, 18, or 25 keV and a fluorescing thin crystal plate that is imaged (at selectable magnification) with a lens onto a 2.5 x 2.5-cm, 1,024 x 1,024-pixel, charge-coupled device (CCD) detector array. The specimen is positioned close to the crystal and is rotated in 721 equiangular steps around 360 degrees between each X-ray exposure and its CCD recording. Tomographic reconstruction algorithms, applied to these recorded images, are used to generate 3-D images of the specimen. The system is used to scan isolated, intact, fixed rodent organs (e.g., heart or kidney) with the image contrast of vessel lumens enhanced with contrast medium. 3-D image display and analysis are used to address physiological questions about the internal structure-to-function relationships of the organs.

Coronary vasa vasorum neovascularization precedes epicardial endothelial dysfunction in experimental hypercholesterolemia

OBJECTIVEnExperimental hypercholesterolemia is associated with vasa vasorum neovascularization, unknown to occur before or after initial lesion formation. Thus, this study was performed to determine the temporal course of neovascularization of coronary vasa vasorum in relation to endothelial dysfunction, a hallmark of early atherosclerosis.nnnMETHODSnFemale domestic pigs were fed a normal diet (Group 1), a hypercholesterolemic diet for 2 and 4 weeks (Group 2), or a hypercholesterolemic diet for 6 and 12 weeks (Group 3). In vitro analysis of relaxation response to bradykinin served as an index for epicardial endothelial function. Spatial pattern and density of coronary vasa vasorum were assessed by three-dimensional microscopic computed tomography.nnnRESULTSnRelaxation response of coronary arteries to bradykinin was normal in both Group 1 (93+/-6%) and Group 2 (89+/-7%) but impaired in Group 3 (71+/-11%; P<0.05 vs. Group 1 and 2). In contrast, density of coronary vasa vasorum was significantly higher in both Group 2 (4.88+/-2.45 per-mm(2)) and Group 3 (4.50+/-1.37 per-mm(2)) compared to Group 1 (2.97+/-1.37 per-mm(2); P<0.05 vs. Group 2 and 3).nnnCONCLUSIONnThis study demonstrates that coronary vasa vasorum neovascularization occurs within the first weeks of experimental hypercholesterolemia and prior to the development of endothelial dysfunction of the host vessel, suggesting a role for vasa vasorum neovascularization in the initial stage of atherosclerotic vascular disease.

Adventitial vasa vasorum in balloon-injured coronary arteries: visualization and quantitation by a microscopic three-dimensional computed tomography technique.

OBJECTIVESnThe objective of this study was to examine the quantitative response of the adventitial vasa vasorum to balloon-induced coronary injury.nnnBACKGROUNDnRecent attention has focused on the role of vasa vasorum in atherosclerotic and restenotic coronary artery disease. However, the three-dimensional anatomy of these complex vessels is largely unknown, especially after angioplasty injury. The purpose of this study was to visualize and quantitate three-dimensional spatial patterns of vasa vasorum in normal and balloon injured porcine coronary arteries. We also studied the spatial growth of vasa vasorum in regions of neointimal formation. A novel imaging technique, microscopic computed tomography, was used for these studies.nnnMETHODSnFour pigs were killed 28 d after coronary balloon injury, and four pigs with uninjured coronary arteries served as normal controls. The coronary arteries were injected with a low-viscosity, radiopaque liquid polymer compound. Normal and injured coronary segments were scanned using a microscopic computed tomography technique. Three-dimensional reconstructed maximum intensity projection and voxel gradient shading images were displayed at different angles and voxel threshold values, using image analysis software. For quantitation, seven to 10 cross-sectional images (40 normal and 32 balloon injured cross-sections) were captured from each specimen at a voxel size of 21 microm.nnnRESULTSnNormal vasa vasorum originated from the coronary artery lumen, principally at large branch points. Two different types of vasa were found and classified as first-order or second-order according to location and direction. In balloon-injured coronary arteries, adventitial vasa vasorum density was increased (3.16+/-0.17/mm2 vs. 1.90+/-0.06/mm2, p = 0.0001; respectively), suggesting neovascularization by 28 d after vessel injury. Also, in these injured arteries, the vasa spatial distribution was disrupted compared with normal vessels, with proportionally more second-order vasa vasorum. The diameters of first-order and second-order vasa were smaller in normal compared with balloon-treated coronary arteries (p = 0.012 first-order; p < 0.001, second-order; respectively). The density of newly formed vasa vasorum was proportional to vessel stenosis (r = 0.81, p = 0.0001). Although the total number of vasa was increased after injury, the total vascular area comprised of vasa was significantly reduced in injured vessels compared with normals (3.83+/-0.20% to 5.42+/-0.56%, p = 0.0185).nnnCONCLUSIONSnAdventitial neovascularization occurs after balloon injury. The number of new vessels is proportional to vessel stenosis. These findings may hold substantial implications for the therapy of vascular disease and restenosis.

Molecular imaging in small animals--roles for micro-CT.

X‐ray micro‐CT is currently used primarily to generate 3D images of micro‐architecture (and the function that can be deduced from it) and the regional distribution of administered radiopaque indicators, within intact rodent organs or biopsies from large animals and humans. Current use of X‐ray micro‐CT can be extended in three ways to increase the quantitative imaging of molecular transport and accumulation within such specimens. (1) By use of heavy elements, other than the usual iodine, attached to molecules of interest or to surrogates for those molecules. The accumulation of the indicator in the physiological compartments, and the transport to and from such compartments, can be quantitated from the imaged spatial distribution of these contrast agents. (2) The high spatial resolution of conventional X‐ray attenuation‐based CT images can be used to improve the quantitative nature of radionuclide‐based tomographic images (SPECT & PET) by providing correction for attenuation of the emitted gamma rays and the accurate delineation of physiological spaces known to selectively accumulate those indicators. Similarly, other imaging modalities which also localize functions in 2D images (such as histological sections subsequently obtained from the same specimen), can provide a synergistic combination with CT‐based 3D microstructure. (3) By increasing the sensitivity and specificity of X‐ray CT image contrast by use of methods such as: K‐edge subtraction imaging, X‐ray fluorescence imaging, imaging of the various types of scattered X‐ray and the consequences of the change in the speed of X‐rays through different tissues, such as refraction and phase shift. These other methods of X‐ray imaging can increase contrast by more than an order of magnitude over that due to conventionally‐used attenuation of X‐ray. To fully exploit their potentials, much development of radiopaque indicators, scanner hardware and image reconstruction and analysis software will be needed. J. Cell. Biochem. Suppl. 39: 116–124, 2002.

Cortical Microvascular Remodeling in the Stenotic Kidney: Role of Increased Oxidative Stress

Objective—Mechanisms of renal injury distal to renal artery stenosis (RAS) remain unclear. We tested the hypothesis that it involves microvascular remodeling consequent to increased oxidative stress. Methods and Results—Three groups of pigs (n=6 each) were studied after 12 weeks of RAS, RAS+antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily), or controls. The spatial density and tortuousity of renal microvessels (<500 &mgr;m) were tomographically determined by 3D microcomputed tomography. The in situ production of superoxide anion and the expression of vascular endothelial growth factor (VEGF), its receptor VEGFR-2, hypoxia-inducible-factor (HIF)-1&agr;, von Hippel-Lindau (VHL) protein, and NAD(P)H oxidase (p47phox and p67phox subunits) were determined in cortical tissue. RAS and RAS+antioxidant groups had similar degrees of stenosis and hypertension. The RAS group showed a decrease in spatial density of cortical microvessels, which was normalized in the RAS+antioxidant group, as was arteriolar tortuousity. RAS kidneys also showed tissue fibrosis (by trichrome and Sirius red staining), increased superoxide anion abundance, NAD(P)H oxidase, VHL protein, and HIF-1&agr; mRNA expression. In contrast, expression of HIF-1&agr;, VEGF, and VEGFR-2 protein was downregulated. These were all significantly improved by antioxidant intervention. Conclusions—Increased oxidative stress in the stenotic kidney alters growth factor activity and plays an important role in renal microvascular remodeling, which can be prevented by chronic antioxidant intervention.

High-speed three-dimensional X-ray computed tomography: The dynamic spatial reconstructor

Most X-ray CT scanners require a few seconds to produce a single two-dimensional (2-D) image of a cross section of the body. The accuracy of full three-dimensional (3-D) images of the body synthesized from a contiguous set of 2-D images produced by sequential CT scanning of adjacent body slices is limited by 1) slice-to-slice registration (positioning of patient); 2) slice thickness; and 3) motion, both voluntary and involuntary, which occurs during the total time required to scan all slices. Therefore, this method is inadequate for true dynamic 3-D imaging of moving organs like the heart, lungs, and circulation. To circumvent these problems, the Dynamic Spatial Reconstructor (DSR) was designed by the Biodynamics Research Unit at the Mayo Clinic to provide synchronous volume imaging, that is stop-action (1/100 s), high-repetition rate (up to 60/s), simultaneous scanning of many parallel thin cross sections (up to 240, each 0.45 mm thick, 0.9 mm apart) spanning the entire anatomic extent of the bodily organ(s)of interest. These capabilities are achieved by using multiple X-ray sources and multiple 2-D fluoroscopic video camera assemblies on a continually rotating gantry. Desired tradeoffs between temporal, spatial, and density resolution can be achieved by retrospective selection and processing of appropriate subsets of the total data recorded during a continuous DSR scan sequence.

Three-dimensional microcomputed tomography of renal vasculature in rats

Current microscopic methods to view renal microvasculature reveal only a very limited portion of the total renal volume. Identification of connectivity for postglomerular vessels in the cortex and the medulla during functional states related to changes in sodium excretion will help better to understand the coupling of renal vasculature to tubular function. The purpose of this study was to investigate the possibility of visualizing the continuity of pre- and postglomerular vasculature using three-dimensional micro-computed tomography (micro-CT). Kidneys from normal rats were perfusion fixed in situ at physiological pressure, filled with latex microfil containing lead chromate, and embedded in plastic. The micro-CT scans of the intact kidneys were carried out on a rotating stage illuminated either by a synchrotron x-ray source or a conventional x-ray spectroscopy tube. Images were reconstructed by a filtered backprojection algorithm and volume-rendering techniques were utilized to display the vasculature. The reconstructed images clearly showed the large distribution vessels and the venous drainage of the kidneys, while pre- and postglomerular vessels and their vascular connections throughout the kidney were displayed in great detail. Efferent arterioles showed the characteristics of their peritubular capillary beds in the cortical and medullary regions. The vascular volume of the cortex was 27%, the outer stripe of the outer medulla 18%, the inner stripe of the outer medulla 30%, and the inner medulla 18%. In conclusion, micro-CT is a promising method to evaluate renal vascular architecture relative to physiological and pathological alterations.

Vascular Endothelial Growth Factor Is Required for Coronary Collateral Growth in the Rat

Background— The goal of this study was to determine whether the expression of vascular endothelial growth factor (VEGF) is critical for coronary collateral growth. Previous studies have provided an association between coronary collateral growth and VEGF, but none have allowed determination of a causal role. Methods and Results— We measured coronary collateral growth in rats subjected to repetitive episodes of myocardial ischemia (RI; one 40-second occlusion every 20 minutes for 2 hours 40 minutes, followed by 5 hours 20 minutes of rest, with this 8-hour cycle repeated 3 times per day for 10 days). Collateral growth was measured from blood flow (radioactive microspheres), visualization of arterial-arterial anastomoses (x-ray micro-CT), and maintenance of function during complete coronary occlusion in 3 groups of animals: sham (received instrumentation but no RI), experimental (subjected to RI), and anti–vascular endothelial growth factor (RI+anti-VEGF 0.6 mg/100 g per day) to block the endogenous actions of VEGF. In the 3 groups, native collateral flow (measurement for RI or sham protocol) averaged 0.2 to 0.3 mL · min−1 · g−1 of tissue. In the sham group, collateral flow did not increase during the protocol. Collateral flow in the control RI group increased by ≈6-fold to 1.63 mL · min−1 · g−1 tissue, but in the anti-VEGF group, collateral flow did not increase after the RI protocol (0.22 mL · min−1 · g−1). In acute experiments, collateral flow was unchanged during vasodilation with dipyridamole, indicating the increases in collateral flow are due to collateral growth and not vasodilation. X-ray micro-CT analysis revealed a 3-fold increase (versus sham group) in the number of arterial-arterial anastomoses per heart after RI, which was prevented by treatment with anti-VEGF. The growth of the collateral circulation was functional in the RI group because complete coronary occlusion did not induce any untoward effects on hemodynamics or arrhythmias. In the sham or anti-VEGF groups, coronary occlusion at the end of the protocol induced many arrhythmias and deterioration of function. Conclusions— From these results, we conclude that the expression of VEGF is critical to the growth of coronary collaterals.

Antioxidant Intervention Attenuates Myocardial Neovascularization in Hypercholesterolemia

Background—Hypercholesterolemia (HC) and atherosclerosis can elicit oxidative stress, coronary endothelial dysfunction, and myocardial ischemia, which may induce growth-factor expression and lead to myocardial neovascularization. We tested the hypothesis that chronic antioxidant intervention in HC would attenuate neovascularization and preserve the expression of hypoxia-inducible factor (HIF)-1&agr; and vascular endothelial growth factor (VEGF). Methods and Results—Three groups of pigs (n= 6 each) were studied after 12 weeks of normal or 2% HC diet or HC+ antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily). Myocardial samples were scanned ex vivo with a novel 3D micro-CT scanner, and the spatial density and tortuosity of myocardial microvessels were determined in situ. VEGF mRNA, protein levels of VEGF and VEGF receptor-1, HIF-1&agr;, nitrotyrosine, and superoxide dismutase (SOD) were determined in myocardial tissue. The HC and HC+ antioxidant groups had similar increases in serum cholesterol levels. HC animals showed an increase in subendocardial spatial density of microvessels compared with normal (160.5± 11.8 versus 95.3± 8.2 vessels/cm2, P < 0.05), which was normalized in HC+ antioxidant (92.5± 20.5 vessels/cm2, P < 0.05 versus HC), as was arteriolar tortuosity. In addition, HC induced upregulation of VEGF, HIF-1&agr;, and nitrotyrosine expression and decreased SOD expression and activity, all of which were preserved by antioxidant intervention. Conclusions—Changes in myocardial microvascular architecture invoked by HC are accompanied by increases in HIF-1&agr; and VEGF expression and attenuated by antioxidant intervention. This underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early atherogenesis.