Martin Rodriguez-Porcel

US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice

PURPOSE To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models. MATERIALS AND METHODS Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance. RESULTS In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types. CONCLUSION US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.

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 F2&agr; (PGF2&agr;)–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, PGF2&agr;-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.

Distinct Renal Injury in Early Atherosclerosis and Renovascular Disease

Background—Atherosclerotic renovascular disease may augment deterioration of renal function and ischemic nephropathy compared with other causes of renal artery stenosis (RAS), but the underlying mechanisms remain unclear. This study was designed to test the hypothesis that concurrent early atherosclerosis and hypoperfusion might have greater early deleterious effects on the function and structure of the stenotic kidney. Methods and Results—Regional renal hemodynamics and function at baseline and during vasoactive challenge (acetylcholine or sodium nitroprusside) were quantified in vivo in pigs by electron-beam computed tomography after a 12-week normal (n=7) or hypercholesterolemic (HC, n=7) diet, RAS (n=6), or concurrent HC and a similar degree of RAS (HC+RAS, n=7). Flash-frozen renal tissue was studied ex vivo. Basal cortical perfusion and single-kidney glomerular filtration rate (GFR) were decreased similarly in the stenotic RAS and HC+RAS kidneys, but tubular fluid reabsorption was markedly impaired only in HC+RAS. Perfusion responses to challenge were similarly blunted in the experimental groups. Stimulated GFR increased in normal, HC, and RAS (38.3±3.6%, 36.4±7.6%, and 60.4±9.3%, respectively, P <0.05), but not in HC+RAS (6.5±15.1%). These functional abnormalities in HC+RAS were accompanied by augmented perivascular, tubulointerstitial, and glomerular fibrosclerosis, inflammation, systemic and tissue oxidative stress, and tubular expression of nuclear factor-&kgr;B and inducible nitric oxide synthase. Conclusions—Early chronic HC+RAS imposes distinct detrimental effects on renal function and structure in vivo and in vitro, evident primarily in the tubular and glomerular compartments. Increased oxidative stress may be involved in the proinflammatory and progrowth changes observed in the stenotic HC+RAS kidney, which might potentially facilitate the clinically observed progression to end-stage renal disease.

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.

Mechanisms of Renal Structural Alterations in Combined Hypercholesterolemia and Renal Artery Stenosis

Objective—Atherosclerotic renovascular disease (ARVD) aggravates renal scarring more than other causes of renal artery stenosis (RAS), but the underlying pathogenic mechanisms of this potential profibrotic effect remain unclear. We tested the hypothesis that coexistence of atherosclerosis and RAS interferes with renal tissue remodeling. Methods and Results—Single-kidney hemodynamics and function were quantified in vivo with electron-beam computed tomography in 3 groups of pigs (n=7 each): normal pigs, pigs 12 weeks after induction of unilateral RAS (RAS group), and pigs with similar-degree RAS fed a 12-week 2% hypercholesterolemic diet (HC+RAS, simulating early ARVD). Kidneys were studied ex vivo by Western blotting and immunohistochemistry. Renal volume, renal blood flow, and glomerular filtration rate were similarly decreased in RAS and HC+RAS ischemic kidneys, accompanied by similar increased expression of profibrotic factors like transforming growth factor-&bgr;, tissue inhibitor of metalloproteinase-1, and plasminogen activator inhibitor-1. Nevertheless, HC+RAS kidneys showed increased intrarenal fibrosis compared with RAS-only kidneys. Furthermore, expression of nuclear factor-&kgr;B was increased, expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems was decreased, and apoptosis was blunted. Conclusions—Diet-induced HC superimposed on RAS accelerates the development of fibrosis in the stenotic kidney by amplifying profibrotic mechanisms and disrupting tissue remodeling. These alterations might contribute to renal disease progression in ARVD and might account for the increased propensity for end-stage renal disease.

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.

Antioxidant Intervention Blunts Renal Injury in Experimental Renovascular Disease

Atherosclerotic renovascular disease (RVD) amplifies damage in a stenotic kidney by inducing pro-inflammatory mechanisms and disrupting tissue remodeling. Oxidative stress is increased in RVD, but its direct contribution to renal injury has not been fully established. The authors hypothesized that chronic antioxidant intervention in RVD would improve renal function and attenuate tissue injury. Single-kidney hemodynamics and function at baseline and during vasoactive challenge were quantified using electron-beam computed tomography in pigs after 12 wk of experimental RVD (simulated by concurrent hypercholesterolemia and renal artery stenosis, n = 7), RVD daily supplemented with antioxidant vitamins C (1 g), and E (100 IU/kg) (RVD+Vitamins, n = 7), or controls (normal, n = 7). Renal tissue was studied ex vivo using Western blot analysis and immunohistochemistry. Basal renal blood flow (RBF) and glomerular filtration rate (GFR) were similarly decreased in the stenotic kidney of both RVD groups. RBF and GFR response to acetylcholine was blunted in RVD, but significantly improved in RVD+Vitamins (P < 0.05 versus RVD). RVD+Vitamins also showed increased renal expression of endothelial nitric oxide synthase (eNOS) and decreased expression of NAD(P)H-oxidase, nitrotyrosine, inducible-NOS, and NF-kappaB, suggesting decreased superoxide abundance and inflammation. Furthermore, decreased expression of pro-fibrotic factors in RVD+Vitamins was accompanied by augmented expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems, resulting in significantly attenuated glomerulosclerosis and renal fibrosis. In conclusion, chronic antioxidant intervention in early experimental RVD improved renal functional responses, enhanced tissue remodeling, and decreased structural injury. This study supports critical pathogenic contribution of increased oxidative stress to renal injury and scarring in RVD and suggests a role for antioxidant strategies in preserving the atherosclerotic and ischemic kidney.

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.

Imaging of VEGF receptor in a rat myocardial infarction model using PET.

Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factors receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject. Methods: In Sprague–Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3). Results: Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 ± 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 ± 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels. Conclusion: We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress.

AIMS To investigate the mechanisms underlying the beneficial effect of hypoxia preconditioning (HPC) on mesenchymal stromal cells (MSCs) and optimize novel non-invasive methods to assess the effect of biological interventions aimed to increased cell survival. MAIN METHODS MSCs from rat femur, with or without HPC, were exposed to hypoxic conditions in cell culture (1% O(2) for 24h) and cell survival (by the LDH release assay and Annexin-V staining) was measured. Oxidant status (conversion of dichloro-fluorescein-DCF- and dihydro-ethidium-DHE-, protein expression of oxidant enzymes) was characterized, together with the mobility pattern of cells under stress. Furthermore, cell survival was assessed non-invasively using state-of-the-art molecular imaging. KEY FINDINGS Compared to controls, Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subunit 67(phox): 0.05 ± 0.01AU and 0.48 ± 0.02AU, respectively, p<0.05) and in the amount of ROS (DCF: 13 ±1 and 42 ± 3 RFU/μg protein, respectively, p<0.05) which led to a decrease in stem cell viability. Hypoxia preconditioning preserved cell biology, as evidenced by preservation of oxidant status (16 ± 1 RFU/μg protein, p<0.05 vs. hypoxia), and cell viability. Most importantly, the beneficial effect of HPC can be assessed non-invasively using molecular imaging. SIGNIFICANCE HPC preserves cell viability and function, in part through preservation of oxidant status, and its effects can be assessed using state-of-the-art molecular imaging. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.