Jill L. Anderson

Thrombolysis and myocardial salvage. Results of clinical trials and the animal paradigm--paradoxic or predictable?

T he era of acute reperfusion has witnessed a dramatic change in the management of acute myocardial infarction in an expanding group of patients who are considered eligible for this form of therapy.1-3 The rationale, based on experimental data obtained in animal models, is logical,4-6 and the results of carefully controlled randomized trials have been shown to meet expectations.1,7-12 The restoration of coronary blood flow does reduce mortality, and the earlier this is achieved, the greater is the benefit. Myocardial salvage, although difficult to quantify, is the logical explanation.

The rat femoral arteriovenous fistula model: increased expression of matrix metalloproteinase-2 and -9 at the venous stenosis.

PURPOSE To determine whether a femoral arteriovenous (AV) fistula model in a rat was feasible and whether there is increased expression of matrix metalloproteinase (MMP)-2 and -9 and the tissue inhibitors of MMPs (TIMPs) at the venous stenosis of the fistula. MATERIALS AND METHODS Fifteen male Sprague-Harley rats weighing 353 g +/- 26 underwent creation of an AV fistula between the left femoral artery and ipsilateral femoral vein, with the contralateral femoral vessels serving as controls. The animals were euthanized at day 14 (n = 5) and day 28 (n = 10) after fistula creation. Zymography and Western blot analysis for TIMP-1 and TIMP-2 were performed at the venous stenosis and in control vessels. Hematoxylin and eosin, Verhoeff-van Gieson, Masson trichrome, and alpha-smooth muscle staining were performed at the stenosis and in controls at day 28 in four animals. The intima/media ratio was determined at day 28. RESULTS By day 14, pro-MMP-2 measurements were 8.13 +/- 1.06 at the venous stenosis and 4.1 +/- 1.33 in controls (P < .05). By day 28, they had increased to 18.95 +/- 4.8 at the stenosis and 12.11 +/- 4.84 in controls (P < .05). By day 14, active MMP-2 measurements were 7.38 +/- 1.25 at the stenosis and 2.31 +/- 1.04 in controls (P < .05). By day 28, they had increased to 12.12 +/- 3.45 at the stenosis and 9.26 +/- 3.97 in controls (P < .05). By day 28, pro-MMP-9 measurements were 11.77 +/- 4.71 at the stenosis and 7.78 +/- 3.49 in controls (P < .05), with no difference at day 14. There was no difference in expression of TIMP-1 and TIMP-2. The average intima/media ratio of the stenosis increased by 28% versus controls, and the neointima was composed of primarily alpha-smooth muscle actin-positive cells. CONCLUSIONS A rat femoral AV fistula model was created with venous stenosis formation characterized by thickened neointima composed of alpha-smooth muscle actin-positive cells compared with controls. At the venous stenosis, there was increased expression of pro-MMP-2 and active MMP-2 by days 14 and 28, with significantly increased expression of pro-MMP-9 by day 28.

In Vivo Vibroacoustography of Large Peripheral Arteries

Objective:Vibroacoustography allows imaging of objects on the basis of their acoustic signal emitted during low-frequency (kHz) vibrations produced by 2 intersecting ultrasound beams at slightly different frequencies. This study tested the feasibility of using vibroacoustography to distinguish between normal and calcified femoral arteries in a pig model. Materials and Methods:Thirteen normal porcine femoral arteries, 7 with experimentally induced arterial calcifications, and 1 control artery injected with saline only were scanned in vivo. Images were obtained at 45 kHz using a 3 MHz confocal transducer. The acoustic emission signal was detected with a hydrophone placed on the animals limb. Images were reconstructed on the basis of the amplitude of the acoustic emission signal. Vessel patency, vessel dimensions, and the extent of calcified plaques were confirmed in vivo by angiography and conventional ultrasound. Excised arteries were reexamined with vibroacoustography, X-ray radiography, and histology. Results:In vivo, vibroacoustography produced high-resolution, speckle-free images with a high level of anatomic detail. Measurements of femoral artery diameter were similar by vibroacoustography and conventional ultrasound (mean difference ± SD, 0.1 ± 0.4 mm). Calcified plaque area measured by different methods was comparable (vibroacoustography, in vivo: 1.0 ± 0.9 cm2; vibroacoustography in vitro: 1.1 ± 0.6 cm2; X-ray radiography: 0.9 ± 0.6 cm2). The reproducibility of measurements was high. Sensitivity and specificity for detecting calcifications were 100% and 86%, respectively, and positive and negative predictive values were 77% and 100%, respectively. Conclusions:Vibroacoustography provides accurate and reproducible measurements of femoral arteries and vascular calcifications in living animals.

Development and preliminary testing of a translational model of hepatocellular carcinoma for MR imaging and interventional oncologic investigations.

PURPOSE To develop a translational rat hepatocellular carcinoma (HCC) disease model for magnetic resonance (MR) imaging and image-guided interventional oncologic investigations. MATERIALS AND METHODS Male rats underwent sham control surgery (n = 6), selective bile duct ligation (SBDL; n = 4), or common bile duct ligation (CBDL; n = 6), with procedure optimization in four rats and N1S1 hepatoma cell injection into two or three sites in the livers of 12 rats. All rats subsequently underwent MR imaging to assess tumor establishment and volume. Mesenteric angiography and percutaneous MR-guided laser ablation of the liver were performed in a subgroup of animals (n = 4). Animal weight and liver test results were monitored. After harvesting, the livers were subjected to gross and microscopic analysis. Tumor volume and laboratory parameters were assessed between ligation groups. RESULTS MR imaging demonstrated hyperintense T2 and hypointense T1 lesions with tumor induction in five of 10 (50.0%), seven of eight (87.5%), and 12 of 12 (100%) sites in the control, SBDL, and CBDL groups, respectively. Tumor volumes differed significantly by group (P < .02). Mesenteric angiography demonstrated an enhancing tumor stain. Clinical and laboratory assessment revealed a significant decrease in weight (P = .01) and albumin level (P < .01) and an increase in total bilirubin level (P = .02) in CBDL rats but not SBDL rats (P = 1.0). Histologic examination showed high-grade HCCs with local and vascular invasion within the context of early fibrosis in CBDL and SBDL rats. MR-guided laser ablation generated a 1-2-cm ablation zone with histologic findings consistent with reversible and irreversible injury. CONCLUSIONS A biologically relevant rat HCC disease model has been developed for MR imaging and preliminary interventional oncologic applications.

Fetuin-A expression in early venous stenosis formation in a porcine model of hemodialysis graft failure.

PURPOSE Because fetuin-A is a cytokine with multifunctional effects on vascular smooth muscle cells and fibroblasts, the authors examined the course of its expression in early venous stenosis formation in a porcine model of chronic renal insufficiency with polytetrafluoroethylene (PTFE) arteriovenous (AV) hemodialysis grafts. MATERIALS AND METHODS Pigs had chronic renal insufficiency created by complete embolization of the left kidney and partial embolization of the right kidney. Twenty-eight days later, PTFE AV grafts were placed from the carotid artery to the ipsilateral jugular vein, and the animals were euthanized 3 days (n = 4), 7 days (n = 4), or 14 days (n = 4) later. Expression of fetuin-A was determined by Western blot analysis of the venous stenosis, control veins, and plasma. Immunohistochemical analysis of the venous stenosis and control vein was performed. Blood urea nitrogen (BUN) and creatinine were measured before embolization and at the time of graft placement. RESULTS The mean BUN and creatinine levels at graft placement were significantly higher than before embolization (P < .05). Severe venous neointimal hyperplasia occurred by day 14 and was characterized by primarily alpha-smooth muscle actin-positive cells. By day 14, fetuin-A levels had increased significantly (P < .05) at the venous stenosis compared with control veins and in the serum compared with measurements before embolization. CONCLUSIONS Significantly increased expression of fetuin-A was observed in early venous stenosis by day 14 and in serum compared with baseline measurements. Understanding the role of fetuin-A in venous neointimal hyperplasia could help in improving outcomes in patients undergoing hemodialysis.

Molecular bioluminescence imaging as a noninvasive tool for monitoring tumor growth and therapeutic response to MRI-guided laser ablation in a rat model of hepatocellular carcinoma

ObjectivesThe objective of this study was to quantitatively compare tumor imaging by magnetic resonance imaging (MRI) and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2-dimensional BLI and 3-dimensional diffuse luminescence tomography (3D DLIT) in an orthotopic rat model of hepatocellular carcinoma. Materials and MethodsThis study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (n = 3) or a heat shock element luciferase reporter (HSE-luc; n = 4) into the liver. All rats underwent MRI to assess tumor establishment and volume and 2-dimensional BLI to assess tumor luminescence at day 7 with subsequent MRI and 2D BLI and 3D DLIT in select animals at days 14 and 21. Magnetic resonance imaging–guided laser ablation of the tumor was performed with preablation and postablation 2D BLI and/or 3D DLIT (n = 2). The tumors underwent histopathologic analysis to assess tumor viability. ResultsThe MRI scans demonstrated hyperintense T2-weighted lesions at 3 of 3 and 4 of 4 sites in the empty vector and HSE-luc rats, respectively. Two-dimensional BLI quantitation demonstrated 23.0-fold higher radiance in the HSE-luc group compared with the empty vector group at day 7 (P < 0.01) and a significant correlation with tumor volume by MRI (r = 0.86; P < 0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. Three-dimensional DLIT quantitation demonstrated better agreement with thepercentage of nonviable tumor by histopathology than did 2D BLI quantitation after the MRI-guided laser ablation. ConclusionsBioluminescence imaging is feasible as a noninvasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of hepatocellular carcinoma.

IVUS Detection of Vasa Vasorum Blood Flow Distribution in Coronary Artery Vessel Wall

There is an increased body of evidence to suggest that the vasa vasorum play a major role in the progression and complications of vulnerable plaque leading to acute coronary syndrome. We propose that detecting changes in the flow in the vascular wall by intravascular ultrasound signals can quantify the presence of vasa vasorum. The results obtained in a porcine model of atherosclerosis suggest that intravascular ultrasound-based estimates of blood flow in the arterial wall can be used in vivo in a clinical research setting to establish the density of vasa vasorum as an indicator of plaque vulnerability.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2–3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Anatomy of hepatic arteriolo-portal venular shunts evaluated by 3D micro-CT imaging

The liver differs from other organs in that two vascular systems deliver its blood - the hepatic artery and the portal vein. However, how the two systems interact is not fully understood. We therefore studied the microvascular geometry of rat liver hepatic artery and portal vein injected with the contrast polymer Microfil(®). Intact isolated rat livers were imaged by micro-CT and anatomic evidence for hepatic arteriolo-portal venular shunts occurring between hepatic artery and portal vein branches was found. Simulations were performed to rule out the possibility of the observed shunts being artifacts resulting from image blurring. In addition, in the case of specimens where only the portal vein was injected, only the portal vein was opacified, whereas in hepatic artery injections, both the hepatic artery and portal vein were opacified. We conclude that mixing of the hepatic artery and portal vein blood can occur proximal to the sinusoidal level, and that the hepatic arteriolo-portal venular shunts may function as a one-way valve-like mechanism, allowing flow only from the hepatic artery to the portal vein (and not the other way around).

Arterial wall perfusion measured with photon counting spectral x-ray CT

Early atherosclerosis changes perfusion of the arterial wall due to localized proliferation of the vasa vasorum. When contrast agent passes through the artery, some enters the vasa vasorum and increases radiopacity of the arterial wall. Technical challenges to detecting changes in vasa vasorum density include the thin arterial wall, partial volume averaging at the arterial lumen/wall interface and calcification within the wall. We used a photon-counting spectral CT scanner to study carotid arteries of anesthetized pigs and micro-CT of these arteries to quantify vasa vasorum density. The left carotid artery wall was injected with autologous blood to stimulate vasa vasorum angiogenesis. The scans were performed at 25-120 keV; the tube-current-time product was 550 mAs. A 60 mL bolus of iodine contrast agent was injected into the femoral vein at 5mL/s. Two seconds post injection, an axial scan was acquired at every 3 s over 60 s (i.e., 20 time points). Each time point acquired 28 contiguous transaxial slices with reconstructed voxels 0.16 x 0.16 x 1 mm3. Regions-of-interest in the outer 2/3 of the arterial wall and in the middle 2/3 of the lumen were drawn and their enhancements plotted versus time. Lumenal CT values peaked several seconds after injection and then returned towards baseline. Arterial wall CT values peaked concurrent to the lumen. The peak arterial wall enhancement in the left carotid arterial wall correlated with increased vasa vasorum density observed in micro-CT images of the isolated arteries.