A Metabolic Intravascular Platform to Study FDG Uptake in Vascular Injury.

PURPOSE: Metabolic alterations underlie many pathophysiological conditions, and their understanding is critical for the development of novel therapies. Although the assessment of metabolic changes in vivo has been historically challenging, recent developments in molecular imaging have allowed us to study novel metabolic research concepts directly in the living subject, bringing us closer to patients. However, in many instances, there is need for sensors that are in close proximity to the organ under investigation, for example to study vascular metabolism. METHODS: In this study, we developed and validated a metabolic detection platform directly in the living subject under an inflammatory condition. The signal collected by a scintillating fiber is amplified using a photomultiplier tube and decodified by an in-house tunable analysis platform. For in vivo testing, we based our experiments on the metabolic characteristics of macrophages, cells closely linked to inflammation and avid for glucose and its analog 18F-fluorodeoxyglucose (18F-FDG). The sensor was validated in New Zealand rabbits, in which inflammation was induced by either a) high cholesterol (HC) diet for 16 weeks or b) vascular balloon endothelial denudation followed by HC diet. RESULTS: There was no difference in weight, hemodynamics, blood pressure, or heart rate between the groups. Vascular inflammation was detected by the metabolic sensor (Inflammation: 0.60 ± 0.03 AU vs. control: 0.48 ± 0.03 AU, p = 0.01), even though no significant inflammation/atherosclerosis was detected by intravascular ultrasound, underscoring the high sensitivity of the system. These findings were confirmed by the presence of macrophages on ex vivo aortic tissue staining. CONCLUSION: In this study, we validated a tunable very sensitive metabolic sensor platform that can be used for the detection of vascular metabolism, such as inflammation. This sensor can be used not only for the detection of macrophage activity but, with alternative probes, it could allow the detection of other pathophysiological processes.

Circulating humanin levels are associated with preserved coronary endothelial function.

Humanin is a small endogenous antiapoptotic peptide, originally identified as protective against Alzheimer's disease, but subsequently also found on human endothelium as well as carotid artery plaques. Endothelial dysfunction is a precursor to the development of atherosclerotic plaques, which are characterized by a highly proinflammatory, reactive oxygen species, and apoptotic milieu. Previous animal studies demonstrated that humanin administration may improve endothelial function. Thus the aim of this study was to test the hypothesis that patients with coronary endothelial dysfunction have reduced systemic levels of humanin. Forty patients undergoing coronary angiography and endothelial function testing were included and subsequently divided into two groups based on coronary blood flow (CBF) response to intracoronary acetylcholine (normal ≥ 50% increase from baseline, n = 20 each). Aortic plasma samples were obtained at the time of catheterization for the analysis of humanin levels and traditional biomarkers of atherosclerosis including C-reactive protein, Lp-Pla(2), and homocysteine. Baseline characteristics were similar in both groups. Patients with coronary endothelial dysfunction (change in CBF = -33 ± 25%) had significantly lower humanin levels (1.3 ± 1.1 vs. 2.2 ± 1.5 ng/ml, P = 0.03) compared with those with normal coronary endothelial function (change in CBF = 194 ± 157%). There was a significant and positive correlation between improved CBF and humanin levels (P = 0.0091) not seen with changes in coronary flow reserve (P = 0.76). C-reactive protein, Lp-Pla(2), and homocysteine were not associated with humanin levels. Thus we observed that preserved human coronary endothelial function is uniquely associated with higher systemic humanin levels, introducing a potential diagnostic and/or therapeutic target for patients with coronary endothelial function.

Role of oxidative stress in hypoxia preconditioning of cells transplanted to the myocardium: a molecular imaging study.

AIM: Cell-based therapies are a potential therapeutic alternative for the treatment of coronary artery disease (CAD). However, transplanted cells undergo significant death in the living subject. Hypoxic preconditioning (HPC) is a potential intervention to increase transplanted cell survival. However, the biological mechanisms of this benefit remain unclear. We hypothesize that the beneficial effect of HPC on stem cell survival is in part due to preservation of oxidant status, an effect that will be monitored using state-of-the-art molecular imaging. METHODS: H9c2 rat cardiomyoblasts expressing the construct CMV-firefly luciferase (h9c2-fluc), with and without HPC, were exposed to hypoxia, and oxidative stress and cell survival were measured. Subsequently, H9c2-fluc cells, with and without HPC, were injected into the myocardium of rats and cell survival was monitored daily with Bioluminescence (BLI) using a CCD camera. RESULTS: Compared to controls, cells exposed to hypoxia had increased amount of reactive oxygen species (ROS, control: 14.1±0.9 vs. hypoxia: 19.5 ± 2.0 RFU/µg protein, P=0.02) and decreased cell survival (control: 0.29 ± 0.005 vs. hypoxia: 0.24 ± 0.005 OD, P<0.001). HPC treatment decreased the amount of hypoxia-induced ROS (HPC: 11.5 ± 0.7RFU/µg protein, P=0.002 vs. hypoxia and P=0.11 vs. control), associated with improved survival (HPC: 0.27 ± 0.004OD/µg protein, P=0.002 vs. hypoxia and P=0.005 vs. control). Most importantly, compared to un-conditioned cells, HPC-cells had increased cell survival after transplantation to the myocardium (C: 34.7 ± 6.7% vs. HPC: 83.4 ± 17.5% at day 5 compared to day 1, P=0.01). CONCLUSION: The beneficial effect of HPC is in part due to preservation of oxidant status. Molecular imaging can assess changes in cell survival in the living subject and has the potential to be applied clinically.

Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy.

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

Renal blood flow in hypercholesterolemic pigs is increased by chronic antioxidant treatment.

Oxygen radical species can influence vascular tone, and antioxidants may have hemodynamic and vascular effects. To date, the vascular effects of chronic intervention with a combination of antioxidant vitamins E and C on renal blood flow (RBF) in hypercholesterolemia (which increases oxidative stress) have not been fully defined. The aim of this intervention study was to explore the involvement of increased oxidative stress in pig RBF disturbance by using chronic dietary antioxidant vitamin intervention. Responses of RBF to the acetylcholine (Ach) were measured in vivo using electron beam computed tomography (EBCT). Acetylcholine significantly increased RBF in normal and hypercholesterolemic + vitamins (P < 0.05 for both), but not in hypercholesterolemic pigs (P=0.1). In normocholesterolemic + vitamins pigs, Ach infusion did not induce any further increase in RBF, but RBF was similar to that observed in normal and hypercholesterolemic + vitamins under the same conditions, and tended to be higher than in hypercholesterolemic pigs (P=0.06). Thus, antioxidants improve RBF in hypercholesterolemic pigs and this effect may help to prevent renal diseases and hypertension in animals.

Coronary vasa vasorum neovascularization precedes epicardial endothelial dysfunction in experimental hypercholesterolemia.

OBJECTIVE: Experimental 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. METHODS: Female 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. RESULTS: Relaxation 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). CONCLUSION: This 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.

Functional assessment of the circulation of the single kidney.

Functional alterations in the renal circulation that can contribute to abnormal renal perfusion have been demonstrated in various models of renal injury. To detect impairments in renal vascular function, renal flow reserve can be determined by repeated measurements of renal blood flow (RBF) during pharmacological challenge with short-acting vasodilators that should increase RBF in kidneys that are not severely damaged structurally. Among the invasive techniques for such measurements, the most readily available is probably the intravascular Doppler, which can be employed during renal angiography for rapid evaluation of changes in RBF during intrarenal injections of vasoactive substances. High-resolution tomographic imaging techniques, like electron-beam x-ray computed tomography, further offer the potential for noninvasive measurements of renal parenchymal perfusion and function, in association with either intrarenal or systemic injections of vasoactive substances. Acetylcholine is a potent short-acting renal vasodilator that can be useful to assess the response of the renal microcirculation, define renal flow reserve, and examine the endothelium-dependent responses of RBF. Such assessments of the function of the renal circulation can assist in evaluation of patients with systemic or renal disease for early detection and monitoring of renovascular injury.

Increased oxidative stress in experimental renovascular hypertension.

The pathophysiological mechanisms responsible for maintenance of chronic renovascular hypertension remain undefined. Excess angiotensin II generation may lead to release of reactive oxygen species and increased vasoconstrictor activity. To examine the potential involvement of oxidation-sensitive mechanisms in the pathophysiology of renovascular hypertension, blood samples were collected and renal blood flow measured with electron-beam computed tomography in pigs 5 and 10 weeks after induction of unilateral renal artery stenosis (n=7) or sham operation (n=7). Five weeks after procedure, plasma renin activity and mean arterial pressure were elevated in hypertensive pigs. Levels of prostaglandin F2alpha (PGF(2alpha))-isoprostanes, vasoconstrictors and markers of oxidative stress, also were significantly increased (157+/-21 versus 99+/-16 pg/mL; P<0.05) and correlated with both plasma renin activity (r=0.83) and arterial pressure (r=0.82). By 10 weeks, plasma renin activity returned to baseline but arterial pressure remained elevated (144+/-10 versus 115+/-5 mm Hg; P:<0.05). Isoprostane levels remained high and still correlated directly with the increase in arterial pressure (r=0.7) but not with plasma renin activity. Stenotic kidney blood flow was decreased at both studies. In shock-frozen cortical tissue, ex vivo endogenous intracellular radical scavengers were significantly decreased in both kidneys. The present study demonstrates, for the first time, that in early renovascular hypertension, an increase in plasma renin activity and arterial pressure is associated with increased systemic oxidative stress. When plasma renin activity later declines, PGF(2alpha)-isoprostanes remain elevated, possibly due to local activation or slow responses to angiotensin II, and may participate in sustenance of arterial pressure. Moreover, oxidation-sensitive mechanisms may influence ischemic and hypertensive parenchymal renal injury.

Combination of hypercholesterolemia and hypertension augments renal function abnormalities.

Hypercholesterolemia and hypertension are both risk factors for end-stage renal disease. This study was designed to examine whether their coexistence augmented impairment in renal function and redox status. Regional renal hemodynamics and function in response to vasoactive challenges with acetylcholine or sodium nitroprusside were quantified by using electron-beam computed tomography in pigs after 12 weeks of either a normal (n=10) or hypercholesterolemic (n=10) diet, renovascular hypertension (n=7), or combined hypercholesterolemia+hypertension (n=6). The hypercholesterolemic and hypercholesterolemic+hypertensive groups had significantly increased serum cholesterol levels, whereas in the hypertensive and hypercholesterolemic+hypertensive groups, mean arterial pressure was significantly elevated compared with the group fed a normal diet. Basal regional renal perfusion and glomerular filtration rates were similar among the groups. In response to acetylcholine, cortical perfusion increased in normal animals (15.6+/-4.7%, P=0.002) but not in hypercholesterolemic or hypertensive animals (8.0+/-7.4% and 8.2+/-5.9%, respectively; P>0.05). Moreover, in the hypercholesterolemic+hypertensive group, cortical perfusion response was further attenuated (2.5+/-4.8%, P=0.02) and significantly different from the group fed a normal diet (P<0.05). The response to sodium nitroprusside followed a similar pattern, and the impairment was augmented in the hypercholesterolemic+hypertensive group. The functional abnormalities in hypercholesterolemia or hypertension were associated with a decrease in systemic and/or renal tissue levels of oxygen radical scavengers that was again accentuated in hypercholesterolemia+hypertension. These results demonstrate that concurrent hypercholesterolemia and hypertension have a greater detrimental effect on renal perfusion responses compared with hypercholesterolemia or hypertension alone, associated with a marked pro-oxidant shift in redox status. These effects may potentially augment renal functional impairment and play a role in the initiation and progression of renal injury in hypertension and atherosclerosis.

c-myc activation in early coronary lesions in experimental hypercholesterolemia.

This study tested the hypothesis that c-Myc activation, an oxidation-sensitive transcription factor, and its binding partner Max occurs in coronary arteries of hypercholesterolemic (HC) pigs, and can be attenuated by chronic antioxidant intervention. Coronary arteries were isolated from normal, HC pigs, or HC supplemented with antioxidant vitamins (HC + vitamins). The expression of the c-Myc/Max complex, and its target genes GADD45 and p53, was studied in nonatherosclerotic, early lesions (LL), positively staining for oil-red-O, in adjacent lesion-prone regions (PL), and in healthy segments (HV). The expression of c-Myc and Max in HC was 2- to 3-fold greater in PL, and 4-fold in LL, compared to normal vessels (P < 0.01). The expression of GADD45 was down-regulated, and of p53 increased, in the same regions. These alterations were attenuated in the HC + vitamins. Thus, c-Myc activation is an early atherosclerosis, in both PL and LL coronary arterial regions, and can be blunted by chronic dietary antioxidant intervention.

Hypercholesterolemia impairs myocardial perfusion and permeability: role of oxidative stress and endogenous scavenging activity.

OBJECTIVES: We intended to study the effect of hypercholesterolemia (HC) on myocardial perfusion and permeability response to increased cardiac demand. BACKGROUND: Hypercholesterolemia is associated with increased incidence of cardiac events and characterized by impaired coronary vascular function, possibly mediated partly through increased pro-oxidative conditions in plasma and tissue. However, it is yet unclear whether HC is also associated with impaired myocardial perfusion and vascular permeability responses in vivo. METHODS: For 12 weeks pigs were fed a normal, HC or HC diet supplemented daily with antioxidants (HC + AO, 100 IU/kg vitamin E and 1 g vitamin C). Myocardial perfusion and vascular permeability were measured in vivo using electron beam computed tomography before and after cardiac challenge with intravenous adenosine. Plasma and tissue oxidative status was determined ex vivo. RESULTS: Plasma cholesterol increased in all cholesterol-fed pigs but was associated with increased markers of oxidative stress only in HC pigs. Myocardial perfusion increased in response to adenosine in normal and HC + AO (+37 +/- 13% and +58 +/- 22%, respectively, p < 0.05 vs. baseline) but not in HC, whereas vascular permeability index increased only in HC pigs (+ 92 +/- 25%, p = 0.002). In HC animals, tissue endogenous oxygen radical scavengers and antioxidant vitamins were depleted and LDL oxidizability enhanced, but both were normalized in HC + AO pigs. Myocardial perfusion response was directly, and permeability inversely, associated with plasma and tissue vitamin concentrations. CONCLUSIONS: This study demonstrates that experimental HC is associated with blunted myocardial perfusion and increased vascular permeability responses in vivo to increased cardiac demand, which may be partly mediated by a shift in oxidative status.

Impaired renal vascular endothelial function in vitro in experimental hypercholesterolemia.

Hypercholesterolemia (HC) induces alterations in systemic vascular reactivity, which can manifest as an attenuated endothelium-dependent relaxation, partly consequent to an impairment in nitric oxide (NO) activity. To determine whether experimental HC has a similar effect on renal vascular function, renal artery segments obtained from pigs fed a HC (n=5) or normal (n=5) diet were studied in vitro. Endothelium-dependent relaxation was examined using increasing concentrations of acetylcholine (Ach), calcium ionophore A23187, and Ach following pre-incubation with N(G)-monomethyl-L-arginine or L-arginine (L-ARG). The NO-donor diethylamine (DEA) was used to examine smooth muscle relaxation response and cyclic GMP generation in endothelium-denuded vessels. The expression of endothelial NO synthase (eNOS) in the renal arteries was examined using Western blotting. Endothelium-dependent relaxation to Ach was significantly attenuated in the HC group compared to normal (53.3+/-9.1 vs. 98.8+/-3.7%, P<0.005), but normalized after pre-incubation with L-ARG (82.3+/-13.8%, P=0.21). Receptor-independent endothelium-dependent relaxation to A23187 was also significantly blunted in HC (75.2+/-10.5 vs. 115.5+/-4.2%, P<0. 017). Smooth muscle relaxation and cyclic GMP generation in response to DEA were greater in denuded HC vessels, while relaxation of intact vessels to nitroprusside was unaltered. In the HC vessels eNOS was almost undetectable. In conclusion, experimental HC attenuates in vitro endothelium-dependent relaxation of the porcine renal artery, possibly due to low bioavailability of NO. These vascular alterations in HC could play a role in the pathogenesis of renal disease or hypertension, supporting a role for HC as a risk factor for renovascular disease.

Altered myocardial microvascular 3D architecture in experimental hypercholesterolemia.

BACKGROUND: Experimental hypercholesterolemia (HC) impairs intramyocardial microvascular function. However, whether this is associated with alterations in microvascular architecture remained unknown. Using a novel 3D micro-CT scanner, we tested the hypothesis that HC is associated with an alteration in the microvascular architecture. METHODS AND RESULTS: Pigs were euthanized after 12 weeks of either normal (n=6) or 2% HC (n=6) diet. The hearts were excised and the coronary arteries injected with a radiopaque contrast material. Myocardial samples were scanned with micro-CT, and 3D images were reconstructed with 21-microm cubic voxels. The myocardium was tomographically subdivided into subepicardium and subendocardium, and microvessels (<500 microm in diameter) were counted in situ within each region. In the subendocardium of HC pigs, the intramyocardial density of microvessels was significantly higher than in normal animals (1221.4+/-199.7 versus 758.3+/-90.8 vessels/cm(3), P:<0.05) because of an increase in the number of microvessels <200 microm in diameter (1214.4+/-199.7 versus 746. 6+/-101.5 vessels/cm(3), P:<0.05). The subepicardial vascular density was similar in both groups. CONCLUSIONS: -HC has differential effects on the spatial density of the subendocardial microvasculature that may play a role in regulation and/or spatial distribution of myocardial blood flow. This study also demonstrates the feasibility of studying myocardial microvascular architecture with micro-CT in pathophysiological states.

The development of x-ray imaging to study renal function.

The well-established role of the kidney in control of blood volume and ultimately arterial blood pressure has been underscored by the demonstration of alterations in renal hemodynamics and function recognized as responsible for these and other regulatory mechanisms. Nevertheless, the spatial complexity of intrarenal structure and function has made evident the need to study these separately in different regions of the intact kidney. Because of the introduction of x-rays, assessment of renal function has indeed been one of their attractive applications. However, despite the appeal of their noninvasiveness, several limitations confounded the different x-ray techniques used, most of which remained unresolved until the development of computed tomography. Furthermore, the development of fast imaging, which allows repetitive analysis of the same region of interest during the transit of contrast medium, holds a great potential to estimate intrarenal distribution of blood flow and the dynamic characteristics of tubular fluid flow in individual nephron segments. This latter assessment requires the administration of filterable x-ray contrast medium, which is cleared from the plasma almost exclusively by glomerular filtration, and the generation of contrast dilution curves. A historical review of the development and progress of the various x-ray techniques used will help understand the past and present of x-ray imaging, and will make it easier to envision the importance of their future roles in the study of renal physiology and pathophysiology.

Perfusion pressure dependency of in vivo renal tubular dynamics.

To examine whether changes in renal perfusion pressure (RPP) within the range of autoregulation induce detectable changes in tubular dynamics in an entire nephron population of the intact kidney, we measured, using electron beam computed tomography (EBCT), transit times (TT, s) and intratubular concentration (%) of filterable contrast media in various nephron segments simultaneously with renal regional perfusion. In seven dogs (group A) this was performed at the upper and lower limits of autoregulation (RPP = 130 and 95 mmHg, respectively) while group B (n = 5) served as control. In group A alone, a decrease in RPP led to an increase in TT by 40%, 68%, and 32% in the proximal tubules, ascending limb of Henle's loop, and distal tubules, respectively, in association with an increase in intratubular concentration (+ 50%, 80%, and 42%, respectively). Papillary perfusion decreased, whereas perfusion of the adjacent, outlying inner medulla increased. The decrease in papillary perfusion correlated positively with the concurrent change in sodium excretion (R = 0.81). This study demonstrates that changes in RPP within the autoregulatory range elicit changes of tubular sodium reabsorption mainly in proximal, distal, and ascending tubules, in which most of the nephrons participate. These tubular changes are associated with an alteration of perfusion circumscribed to two areas of the inner renal medulla.

New insights into the pathophysiology of renovascular hypertension.

In recent years, the pathophysiology of renovascular hypertension has been reviewed, and the classic concept that activation of the renin-angiotensin system is solely responsible for the development and maintenance of renovascular hypertension has been challenged. In fact, experimental evidence indicates that other systems, such as the lipoxygenase pathway, may have a more critical role in the long-term maintenance of high blood pressure after renal artery stenosis. Herein we discuss the intrarenal mechanisms that control pressure-induced natriuresis under physiologic conditions and the role of the kidney in the pathophysiology of renovascular hypertension.

Blood pressure monitor-induced petechiae and ecchymoses.

A case is presented of a diabetic, hypertensive, female patient who suffers from a bleeding complication from application of an ambulatory blood pressure monitor. A recent literature search is referred to and practitioners are cautioned against this adverse reaction.