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.

Atherosclerosis and its consequences remain the main cause of mortality in industrialized and developing nations. Plaque burden and progression have been shown to be independent predictors for future cardiac events by intravascular ultrasound. Routine prospective imaging is hampered by the invasive nature of intravascular ultrasound. A noninvasive technique would therefore be more suitable for screening of atherosclerosis in large populations. Here we introduce an elastin-specific magnetic resonance contrast agent (ESMA) for noninvasive quantification of plaque burden in a mouse model of atherosclerosis. The strong signal provided by ESMA allows for imaging with high spatial resolution, resulting in accurate assessment of plaque burden. Additionally, plaque characterization by quantifying intraplaque elastin content using signal intensity measurements is possible. Changes in elastin content and the high abundance of elastin during plaque development, in combination with the imaging properties of ESMA, provide potential for noninvasive assessment of plaque burden by molecular magnetic resonance imaging (MRI).

The manganese(II) ion has many favorable properties that lead to its potential use as an MRI contrast agent: high spin number, long electronic relaxation time, labile water exchange. The present work describes the design, synthesis, and evaluation of a novel Mn(II) complex (MnL1) based on EDTA and also contains a moiety that noncovalently binds the complex to serum albumin, the same moiety used in the gadolinium based contrast agent MS-325. Ultrafiltration albumin binding measurements (0.1 mM, pH 7.4, 37 degrees C) indicated that the complex binds well to plasma proteins (rabbit: 96 +/- 2% bound, human: 93 +/- 2% bound), and most likely to serum albumin (rabbit: 89 +/- 2% bound, human 98 +/- 2% bound). Observed relaxivities (+/- 5%) of the complex were measured (20 MHz, 37 degrees C, 0.1 mM, pH 7.4) in HEPES buffer (r(1) = 5.8 mM(-)(1) s(-)(1)), rabbit plasma (r(1) = 51 mM(-)(1) s(-)(1)), human plasma (r(1) = 46 mM(-)(1) s(-)(1)), 4.5% rabbit serum albumin (r(1) = 47 mM(-)(1) s(-)(1)), and 4.5% human serum albumin (r(1) = 48 mM(-)(1) s(-)(1)). The water exchange rate was near optimal for an MRI contrast agent (k(298) = 2.3 +/- 0.9 x 10(8) s(-)(1)). Variable temperature NMRD profiles indicated that the high relaxivity was due to slow tumbling of the albumin-bound complex and fast exchange of the inner sphere water. The concept of a high relaxivity Mn(II)-based contrast agent was validated by imaging at 1.5 T. In a rabbit model of carotid artery injury, MnL1 clearly delineated both arteries and veins while also distinguishing between healthy tissue and regions of vessel damage.

Background-The advent of fibrin-binding molecular magnetic resonance (MR) contrast agents and advances in coronary MRI techniques offers the potential for direct imaging of coronary thrombosis. We tested the feasibility of this approach using a gadolinium (Gd)-based fibrin-binding contrast agent, EP-2104R (EPIX Medical Inc), in a swine model of coronary thrombus and in-stent thrombosis. Methods and Results-Ex vivo and in vivo sensitivity of coronary MR thrombus imaging was tested by use of intracoronarily delivered Gd-DTPA-labeled fibrinogen thrombi (nϭ6). After successful demonstration, in-stent coronary thrombosis was induced by x-ray-guided placement of thrombogenic-coated, MR-lucent stents (nϭ5). After stent placement, 60 mol of EP-2104R was injected via the left main coronary artery. Free-breathing, navigator-gated 3D coronary MR angiography and thrombus imaging were performed (1) before and after stent placement and (2) before and after EP-2104R. Thrombi were confirmed by x-ray angiography and autopsy. Fibrinogen thrombi: 5 of 6 intracoronarily delivered Gd-labeled fibrinogen clots (Ϸ250 mol/L Gd) were visible on MRI and subsequently confirmed by x-ray angiography. In-stent thrombi: in-stent thrombosis was observed in all stents after EP-2104R. Four of 5 thrombi were confirmed by x-ray angiography. Chemical analysis of 2 thrombi demonstrated 99 to 147 mol/L Gd. Conclusions-We demonstrate the feasibility of MRI of coronary thrombus and in-stent thrombosis using a novel fibrin-binding molecular MR contrast agent. Potential applications include detection of coronary in-stent thrombosis or thrombus burden in patients with acute coronary syndromes.

This study was an initial phase II trial in humans of molecular magnetic resonance (MR) imaging for improved visualization of thrombi in vessel territories potentially responsible for stroke using a new fibrin-specific contrast agent (EP-2104R). Eleven patients with thrombus in the left ventricle (n = 2), left or right atrium (n = 4), thoracic aorta (n = 4) or carotid artery (n = 1) as verified by an index examination (ultrasound, computed tomograpy, or conventional MR) were enrolled. All MR imaging was performed on 1.5 T whole-body MR-system using an inversion-recovery black-blood gradient-echo sequence. The same sequence was performed before and 2-6 h after low-dose intravenous administration of 4 mumol/kg EP-2104R. Two investigators assessed image quality and signal amplification. Furthermore, contrast-to-noise ratios (CNR) between the clot and the blood pool/surrounding soft tissue before and after administration of the contrast agent were compared using Student's t-test. MR imaging and data analysis were successfully completed in 10 patients. No major adverse effects occurred. On enhanced images, thrombi demonstrated high signal amplification, typically at the clot surface, with a significantly increased contrast in comparison to the surrounding blood pool and soft tissue (CNR for clot vs. blood pool, unenhanced and enhanced: 6 +/- 8 and 29 +/- 14; CNR for clot vs. soft tissue, unenhanced and enhanced: 0 +/- 4 and 21 +/- 13; P < 0.01 for both comparisons). EP-2104R allows for molecular MR imaging of thrombi potentially responsible for stroke. High contrast between thrombus and surrounding blood and soft tissues can be achieved with enhanced imaging.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.Previous studies have demonstrated a cytoprotective function for heat shock proteins during thermal stress or energy deprivation in cultured cells (1-6). Expression of endogenous 70-kDa heat shock protein (hsp7o) is induced by ischemia in intact hearts (6, 7), and this response correlates with a reduced susceptibility to ischemic injury (8-11). Preconditioning stimuli that induce hsp70, however, also invoke other responses that may mitigate myocardial dysfunction resulting from ischemia (12)(13)(14). The proposition that stress proteins exert direct cardioprotective effects remained conjectural until the development of transgenic mouse models, which provide an opportunity for rigorous tests of this hypothesis by examination of relationships between heat shock protein expression and various descriptors of ischemic injury. The goal of this work was to assess the effects of forced constitutive expression of hsp70 on recovery of high-energy phosphate metabolism (assessed by 31P NMR spectroscopy) following periods of global ischemia in isolated perfused mouse hearts. assay. RNA was extracted from myocardial tissue samples by standard methods, and cDNA was synthesized with reverse transcriptase by using an oligo(dT) primer. Primer pairs were specific either for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) or human hsp7o cDNA and generated amplification products of 400 or 496 bp, respectively. RNA isolated from heat shocked human HeLa cells served as a positive control. The identity of the PCR product generated from the transgenic hearts when using human hsp7o primer pairs was confirmed by cleavage with Cla I, the restriction site for which is absent from the murine hsp70 sequence, to produce a 453-bp fragment. Proteins extracted from whole hearts were separated by isoelectric focusing and SDS/PAGE, and two-dimensional immunoblot assays (17) were conducted by using an exquisitely sensitive rabbit antihuman inducible hsp7o antibody. MATERIALS AND METHODSLangendorff Perfusion. The care and treatment of all animals in this study were in accord with the recommendations of the National Institutes of Health and the U. S. Department of Health and Human Services. The protocol was approved by the Institutional Review Board for Animal Rese...

EP-2104R enhanced magnetic resonance imaging detects thrombi not readily visible in precontrast screening and gives additional enhancement of thrombi that are visible in precontrast imaging.

The source of the T1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe3+ form. Quantification of T1 relaxation time appears to be a good predictor of the success of thrombolysis.