William C. Quist

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

In Vivo Molecular Imaging of Acute and Subacute Thrombosis Using a Fibrin-Binding Magnetic Resonance Imaging Contrast Agent

Background—Plaque rupture with subsequent thrombosis is recognized as the underlying pathophysiology of most acute coronary syndromes and stroke. Thus, direct thrombus visualization may be beneficial for both diagnosis and guidance of therapy. We sought to test the feasibility of direct imaging of acute and subacute thrombosis using MRI together with a novel fibrin-binding gadolinium-labeled peptide, EP-1873, in an experimental animal model of plaque rupture and thrombosis. Methods and Results—Fifteen male New Zealand White rabbits (weight, ≈3.5 kg) were made atherosclerotic by feeding a high-cholesterol diet after endothelial aortic injury. Plaque rupture was then induced with the use of Russell’s viper venom (RVV) and histamine. Subsequently, MRI of the subrenal aorta was performed before RVV, after RVV, and after EP-1873. Histology was performed on regions suggested by MRI to contain thrombus. Nine rabbits (60%) developed plaque rupture and thrombus, including 25 thrombi visually apparent on MRI as “hot spots” after injection of EP-1873. Histological correlation confirmed all 25 thrombi (100%), with no thrombi seen in the other regions of the aorta. In the remaining 6 rabbits (control) without plaque rupture, no thrombus was observed on the MR images or on histology. Conclusions—We demonstrate the feasibility of in vivo “molecular” MRI for the detection of acute and subacute thrombosis using a novel fibrin-binding MRI contrast agent in an animal model of atherosclerosis and acute/subacute thrombosis. Potential clinical applications include thrombus detection in acute coronary syndromes and stroke.

Downstream anastomotic hyperplasia. A mechanism of failure in Dacron arterial grafts.

: The precise location and progression of anastomotic hyperplasia and its possible relationship to flow disturbances was investigated in femoro-femoral Dacron grafts in 28 dogs. In 13 grafts, the outflow from the end-to-side downstream anastomosis was bidirectional (BDO), and in 15 it was unidirectional (UDO) (distally). Grafts were electively removed at intervals of two to 196 days or at the time of thrombosis. Each anastomosis and adjacent artery was perfusion-fixed and sectioned sagittally. The mean sagittal section was projected onto a digitized pad, and the total area of hyperplasia internal to the arterial internal elastic lamina and within the adjacent graft was integrated by computer. The location of the hyperplasia was compared with previously established sites of flow separation and stagnation. The observation was made that hyperplasia is significantly greater at the downstream, as compared with the upstream, anastomosis in both groups (BDO = p less than 0.001 and UDO = p less than 0.001) (analysis of variance for independent groups). Furthermore, this downstream hyperplasia was progressive with time (BDO p less than 0.01) (UDO p less than 0.01); Spearman Rank Correlation. There was no significant increase in the extent of downstream hyperplasia where flow separation was known to be greater (BDO). Five grafts failed (three BDO, two UDO), as a result of complete occlusion of the downstream anastomosis by fibrous hyperplasia. Transmission electron microscopy showed the hyperplasia to consist of collagen-producing smooth muscle cells. Anastomotic hyperplasia is significantly greater at the downstream anastomosis, is progressive with time, and is the primary cause of failure of Dacron arterial grafts in this model. Quantitative analysis of downstream anastomotic hyperplasia may be a valuable measure of the biocompatibility of Dacron grafts.

Poly(ADP-Ribose) Polymerase Is Activated in Subjects at Risk of Developing Type 2 Diabetes and Is Associated With Impaired Vascular Reactivity

Background—We have previously shown that endothelial function is impaired not only in diabetes but also in subjects at risk of developing type 2 diabetes. We hypothesized that changes in the expression or activity of the endothelial isoform of nitric oxide synthase (eNOS), the receptor for advanced glycation end products (RAGE), and poly(ADP-ribose) polymerase (PARP) are related to this impairment. Methods and Results—We included a control group of 21 healthy subjects, a group of 22 healthy individuals with parental history of type 2 diabetes, a group of 23 subjects with impaired glucose tolerance, and a group of 21 type 2 diabetic patients. Two 2-mm forearm skin biopsies were taken from each participant and used for measurements. The percentage of PARP-positive endothelial nuclei was higher in the group with parental history of type 2 diabetes and diabetic patients compared with the controls (P <0.001). Immunoreactivity for nitrotyrosine (a marker of reactive nitrogen species) was higher in the diabetic group compared with all other groups (P <0.01). No differences in the expression of eNOS and RAGE were found among all 4 groups. The polymorphism of the eNOS gene was also studied and was not found to influence eNOS expression or microvascular functional measurements. Conclusions—PARP activation is present in healthy subjects at risk of developing diabetes as well as in established type 2 diabetic patients, and it is associated with impairments in the vascular reactivity in the skin microcirculation.

Angiotensin-Converting Enzyme Inhibition Suppresses Plasminogen Activator Inhibitor-1 Expression in the Neointima of Balloon-Injured Rat Aorta

BACKGROUND Plasminogen activator inhibitor-1 (PAI-1), an important regulator of fibrinolysis and extracellular matrix turnover, has been implicated in a number of vascular diseases. Studies demonstrating angiotensin II (Ang II) to be a potent stimulator of PAI-1 expression in cultured vascular cells suggests that the renin-angiotensin system may modulate vascular PAI-1 expression. METHODS AND RESULTS We examined the effects of the ACE inhibitor captopril on PAI-1 expression in control and balloon-injured rat aorta. Northern blot analysis demonstrated that aortic PAI-1 mRNA expression was 7.6-fold elevated 3 hours (P<.05) after balloon injury, back to baseline at 2 days, increased again at 4 days, and by 7 days after balloon injury was 3.2-fold elevated (P<.05) when compared with control. In captopril-treated rats, the induction of PAI-1 expression by balloon injury was significantly suppressed by 44% (P<.05) in the 7 day group but was not altered in the 3-hour group. Captopril also reduced baseline aortic PAI-1 mRNA. In situ hybridization and immunohistochemistry revealed dense PAI-1 staining of 7-day neointima in untreated rats and a dramatic decrease in PAI-1 in neointima of captopril-treated rats. CONCLUSIONS This report demonstrates that balloon injury results in both a rapid ACE inhibitor-independent induction of aortic PAI-1 expression and a later increase in PAI-1 in the neointima that is significantly suppressed by captopril. This provides the first evidence that the renin-angiotensin system regulates neointimal PAI-1 expression and that ACE inhibitors can reduce PAI-1 in the vessel wall in vivo.

Angiotensin Receptor Blockade With Candesartan Attenuates Atherosclerosis, Plaque Disruption, and Macrophage Accumulation Within the Plaque in a Rabbit Model

Background—Little is known about whether direct angiotensin receptor blockade can reduce atherosclerosis and plaque disruption. This study evaluated the effect of angiotensin receptor blockade on both the development of atherosclerosis and the disruption of plaque in a modified Constantinides animal model. Methods and Results—Twenty-eight New Zealand White rabbits underwent aortic balloon injury followed by a 1% cholesterol diet for 8 weeks. Thirteen rabbits received candesartan at 0.5 mg · kg−1 · d−1 beginning 2 days before aortic balloon injury and continued for the total 8 weeks of the cholesterol diet. The rabbits were then pharmacologically triggered and humanely killed, and their aortas were analyzed. The degree of atherosclerosis was determined by intima-media ratio of the infrarenal portion of the aorta. The frequency of intra-aortic thrombosis, a measure of plaque disruption, and the percentages of macrophage area and collagen-staining area of the plaque were determined. Candesartan-treated rabbits had less atherosclerosis (intima-media infrarenal aorta ratio of 1.18±0.08 versus 1.57±0.08 [mean±SEM] for the placebo group, P<0.001); fewer thrombi (3 of 13 versus 11 of 15; P<0.05); lower percentage area of macrophages to total plaque (18.8±2.7% versus 27±2.5%, P<0.05); and higher collagen to total plaque area (45±3% versus 35±2%, P<0.01). Conclusions—These results demonstrate that angiotensin receptor blockade attenuates the degree of atherosclerosis and reduces both plaque disruption and macrophage accumulation while increasing collagen deposition in the aortas of this animal model.

In Vivo Assessment of a Novel Dacron Surface with Covalently Bound Recombinant Hirudin

Prosthetic arterial graft surfaces are relatively thrombogenic and fail to heal with a cellular neointima. The goal of this study was to characterize the in vivo antithrombin properties of a novel Dacron surface with covalently linked recombinant hirudin (rHir) implanted in a canine thoracic aorta with high flow and shear rates. rHir was bound to a knitted Dacron patch using crosslinker-modified bovine serum albumin (BSA) as a basecoat protein. BSA was first reacted with the heterobifunctional crosslinker, sulfo-SMCC. This BSA-SMCC complex was then bound to the carboxylic acid groups of hydrolyzed Dacron patches using the carbodiimide crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Iodinated, Trauts-modified rHir (125I-rHir-SH) was then reacted with the Dacron-BSA-SMCC surface, thereby covalently binding 125I-rHir. Graft segments were washed and sonicated to remove any nonspecifically bound 125I-rHir. Dacron-BSA-SMCC-S-125I-rHir patches (n = 5) and control Dacron-BSA patches (n = 5) were implanted in series in the thoracic aortas of canines. These patches were exposed to nonheparinized, arterial blood flow for 2 hours. Patches were explanted and assessed for 125I-rHir loss. Antithrombin activity of explanted 1-cm2 patch segments was evaluated using a chromogenic assay with 1, 5, 10, 15 units of added thrombin. Light microscopy was performed to qualitatively examine the pseudointima. Two animals were excluded from the study owing to excessive bleeding through the knitted 125I-rHir patch. Comparison of preoperative and postoperative 125I-rHir gamma counts revealed an overall decrease of 20+/-5.4% over the period studied. Explanted 125I-rHir patch segments were able to inhibit 1, 5, and 7 NIHU of thrombin, demonstrating retained antithrombin activity. Gross and microscopic examination of the control and test Dacron surfaces showed marked differences. Dacron surfaces with covalently bound 125I-rHir had no gross thrombus and a thin pseudointima of platelets and plasma proteins. In contrast, the control patches had a thick pseudointima composed of fibrin-rich thrombus. rHir, covalently bound to Dacron patches, maintains its biologic activity as well as prevents thrombus formation on the graft surface. This novel antithrombin coating, by modifying the blood/ graft interface, may improve both short- and long-term patency in small-diameter prosthetic arterial grafts and has applications with respect to other implantable or indwelling biomaterials.

Preclinical safety testing of percutaneous transatrial access to the normal pericardial space for local cardiac drug delivery and diagnostic sampling.

The safety of a percutaneous method and streamlined catheter system to access the normal pericardial space via the right atrial appendage for drug delivery and diagnostic sampling was demonstrated in 20 anesthetized pigs. Access was successfully accomplished in all animals within 3 min of guide catheter positioning and was documented by fluoroscopic imaging and pericardial fluid sampling. The animals were sacrificed at 24 hr (n = 10) and 2 weeks (n = 10) for histopathologic analysis. Mean pericardial hematocrit was 1.1% ± 0.3% at initial sampling, 4.3% ± 1.4% at 24 hr (P = 0.005 vs. baseline), and 0.4% ± 0.2% at 2 weeks (P = 0.13 vs. baseline). At 24 hr, there was local inflammatory reaction in the atrial wall and a small thrombus at the site of puncture. At 2 weeks, no significant inflammatory changes or pericarditis were evident. The technique is well tolerated with no apparent adverse complications. Advances in intrapericardial therapeutics and diagnostics will direct the clinical application of this novel approach in human subjects. Cathet. Cardiovasc. Intervent. 49:472–477, 2000.

Structural details of boundary layer separation in a model human carotid bifurcation under steady and pulsatile flow conditions

We have used a dye-flow visualization technique to analyze the substructure of flow separation in a plastic model of the human carotid bifurcation under steady and pulsatile flow. Under steady conditions at a physiologic flow split (Q external carotid/Q common carotid = 0.30) and Reynolds number (500), a large region of separated fluid developed along the outside wall of the sinus, opposite the flow divider. Yellow dye injected into the boundary layer upstream from the bifurcation traveled slowly along the wall of the common carotid and entered directly into the separation. Blue dye injected into the central, high-velocity streamlines in the common carotid impacted on the flow divider, then traveled circumferentially and entered the separation. Mixing of these two sources was documented by the appearance of green fluid, which lingered in the region of separation. Pulsatile flow resulted in a smaller region of separation; mixing still occurred. Flow separation at the carotid bifurcation is a site of mixing of fluids previously subjected to prolonged low-shear wall contact and brief high-shear wall contact. Separation is itself a site of low shear, but this study reveals a mechanism whereby low and high shear may act independently or synergistically to explain the link between flow separation and atherogenesis.

Vascular endothelial growth factor expression in canine peripheral vein bypass grafts

PURPOSE Autologous veins used as arterial bypass grafts undergo initial loss of the endothelial cell (EC) lining, which is followed by reendothelialization. We characterized the expression of the EC-specific angiogenic mitogen, vascular endothelial growth factor (VEGF), in vascular grafts to help elucidate the molecular and cellular events after bypass procedures. METHODS Cephalic vein-femoral artery interposition grafts were placed in mongrel dogs. Vein grafts and arteries were harvested at either 48 hours or 4 weeks after bypass, the total RNA was isolated, and the VEGF mRNA expression was evaluated by Northern blot analysis. Tissue segments from each time period were evaluated by immunohistochemical analysis using anti-VEGF antibodies. RESULTS VEGF mRNA expression in vein grafts as compared with control veins was increased 2.5-fold 48 hours after bypass grafting (p = 0.02) but returned to initial control levels in grafts removed at 4 weeks. Distal arterial segments, which included the anastomotic site without attached vein graft, had a 21.4-fold increase in VEGF expression at 48 hours (p = 0.02) and a 6.6-fold increase at 4 weeks (p < 0.01) as compared with control arterial segments. Vessels subjected to arteriotomy or ischemia alone also demonstrated increased VEGF expression. Immunohistochemical analysis revealed VEGF protein within ECs and smooth muscle cells of the venous bypass graft, with maximal levels observed within intimal hyperplasia at the arterial anastomosis. CONCLUSIONS After arterial reconstruction procedures using venous conduits, VEGF is significantly increased at 48 hours in the vein graft and arterial anastomosis. VEGF expression in the vein graft normalizes within 4 weeks but remains significantly elevated in the adjacent arterial segment. Increased VEGF production after arterial grafting may facilitate reendothelialization, thus partially accounting for optimal patency rates achieved with autologous vein grafts.