Association between CYP2C9 and VKORC1 genetic polymorphisms and efficacy and safety of warfarin in Chinese patients.

OBJECTIVES: Genetic variation has been a major contributor to interindividual variability of warfarin dosage requirement. The specific genetic factors contributing to warfarin bleeding complications are largely unknown, particularly in Chinese patients. In this study, 896 Chinese patients were enrolled to explore the effect of CYP2C9 and VKORC1 genetic variations on both the efficacy and safety of warfarin therapy. METHODS AND RESULTS: Univariate analyses unveiled significant associations between two specific single nucleotide polymorphisms rs1057910 in CYP2C9 and rs9923231 in VKORC1 and stable warfarin dosage ( P  < 0.001). Further, employing multivariate logistic regression analysis adjusted for age, sex and height, the investigation revealed that patients harboring at least one variant allele in CYP2C9 exhibited a heightened risk of bleeding events compared to those with the wild-type genotype (odds ratio = 2.16, P  = 0.04). Moreover, a meta-analysis conducted to consolidate findings confirmed the associations of both CYP2C9 (rs1057910) and VKORC1 (rs9923231) with stable warfarin dosage. Notably, CYP2C9 variant genotypes were significantly linked to an increased risk of hemorrhagic complications ( P  < 0.00001), VKORC1 did not demonstrate a similar association. CONCLUSION: The associations found between specific genetic variants and both stable warfarin dosage and bleeding risk might be the potential significance of gene detection in optimizing warfarin therapy for improving patient efficacy and safety.

Controlling Reperfusion Injury With Controlled Reperfusion: Historical Perspectives and New Paradigms.

Cardiac reperfusion injury is a well-established outcome following treatment of acute myocardial infarction and other types of ischemic heart conditions. Numerous cardioprotection protocols and therapies have been pursued with success in pre-clinical models. Unfortunately, there has been lack of successful large-scale clinical translation, perhaps in part due to the multiple pathways that reperfusion can contribute to cell death. The search continues for new cardioprotection protocols based on what has been learned from past results. One class of cardioprotection protocols that remain under active investigation is that of controlled reperfusion. This class consists of those approaches that modify, in a controlled manner, the content of the reperfusate or the mechanical properties of the reperfusate (e.g., pressure and flow). This review article first provides a basic overview of the primary pathways to cell death that have the potential to be addressed by various forms of controlled reperfusion, including no-reflow phenomenon, ion imbalances (particularly calcium overload), and oxidative stress. Descriptions of various controlled reperfusion approaches are described, along with summaries of both mechanistic and outcome-oriented studies at the pre-clinical and clinical phases. This review will constrain itself to approaches that modify endogenously-occurring blood components. These approaches include ischemic postconditioning, gentle reperfusion, controlled hypoxic reperfusion, controlled hyperoxic reperfusion, controlled acidotic reperfusion, and controlled ionic reperfusion. This review concludes with a discussion of the limitations of past approaches and how they point to potential directions of investigation for the future.

Acoustic droplet vaporization-mediated dissolved oxygen scavenging in blood-mimicking fluids, plasma, and blood.

Acoustic droplet vaporization (ADV) has been shown to reduce the partial pressure of oxygen (PO2) in a fluid. The goals of this study were three-fold: 1) to determine the ADV pressure amplitude threshold in fluids that had physiologically relevant values for surface tension, protein concentration, and viscosity; 2) to assess whether these parameters and fluid mixing affect ADV-mediated PO2 reduction; and 3) to assess the feasibility of ADV-mediated PO2 reduction in plasma and whole blood. In vitro ADV experiments were conducted using perfluoropentane droplets (number density: 5 × 106 ±â€¯0.2 × 106/mL) dispersed in fluids (saline, polyvinylpyrrolidone solutions, porcine plasma, or porcine whole blood) that had a physiological range of surface tensions (62-68 mN/m), protein concentrations (0 and 68.7 mg/mL), and viscosities (0.7-4 cP). Droplets were exposed to pulsed ultrasound (5 MHz, 4.25 MPa peak negative pressure) while passing through a 37 °C flow system with inline PO2 sensors. In select experiments, the fluid also passed through mixing channels after ultrasound exposure. Our results revealed that the ADV pressure thresholds were the same for all fluids. Surface tension and protein concentration had no effect on PO2 reduction. Increasing viscosity attenuated PO2 reduction. However, the attenuated effect was absent after fluid mixing. Furthermore, ADV-mediated PO2 reduction in whole blood (30.8 ±â€¯3.2 mmHg) was less than that in a polyvinylpyrrolidone solution (40.2 ±â€¯2.1 mmHg) with equal viscosity. These findings should be considered when planning clinical studies of ADV-mediated PO2 reduction and other biomedical applications of ADV.

Dissolved Oxygen Scavenging by Acoustic Droplet Vaporization using Intravascular Ultrasound.

Modification of dissolved gas content by acoustic droplet vaporization (ADV) has been proposed for several therapeutic applications. Reducing dissolved oxygen (DO) during reperfusion of ischemic tissue during coronary interventions could inhibit reactive oxygen species production and rescue myocardium. The objective of this study was to determine whether intravascular ultrasound (IVUS) can trigger ADV and reduce DO. Perfluoropentane emulsions were created using high-speed shaking and microfluidic manufacturing. High-speed shaking resulted in a polydisperse droplet distribution ranging from less than 1 micron to greater than 16 microns in diameter. Microfluidic manufacturing produced a narrower size range of droplets with diameters between 8.0 microns and 9.6 microns. The DO content of the fluids was measured before and after ADV triggered by IVUS exposure. Duplex B-mode and passive cavitation imaging was performed to assess nucleation of ADV. An increase in echogenicity indicative of ADV was observed after exposure with a clinical IVUS system. In a flow phantom, a 20% decrease in DO was measured distal to the IVUS transducer when droplets, formed via high-speed shaking, were infused. In a static fluid system, the DO content was reduced by 11% when droplets manufactured with a microfluidic chip were exposed to IVUS. These results demonstrate that a reduction of DO by ADV is feasible using a clinical IVUS system. Future studies will assess the potential therapeutic efficacy of IVUS-nucleated ADV and methods to increase the magnitude of DO scavenging.

In Vitro and In Vivo Evaluation of Targeted Sunitinib-Loaded Polymer Microbubbles Against Proliferation of Renal Cell Carcinoma.

OBJECTIVES: The poor safety profile of sunitinib capsules has encouraged the identification of targeted drug delivery systems against renal cell carcinoma. This study aimed to explore the effect of sunitinib-loaded microbubbles along with ultrasound (US) treatment on proliferation and apoptosis of human GRC-1 granulocyte renal carcinoma cells in vitro and in vivo (xenograft tumor growth in nude mice). METHODS: Liposomes containing sunitinib were prepared by using the transmembrane ammonium sulfate gradient method and then absorbed into polymer microbubbles to generate sunitinib-loaded microbubbles. Entrapment of sunitinib was verified by 25-25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol staining. GRC-1 cells were treated with microbubbles alone, liposomes alone, sunitinib alone, sunitinib-loaded microbubbles without and with US, and no treatment (control). Cell survival and apoptosis were assessed at 12, 24, and 48 hours after treatment. Xenograft tumors were induced by implantation of GRC-1 cells in nude mice. The animals with tumors were then randomly assigned to sunitinib alone, sunitinib-loaded microbubbles - US, sunitinib-loaded microbubbles + US, and no treatment (control; n = 10 per group). The tumor volumes were analyzed on the 7th, 15th, and 21st days. RESULTS: The sunitinib entrapment efficiency in the liposomes was approximately 78%. The effective sunitinib concentration in each group was 0.1 µg/mL. The sunitinib-loaded microbubble + US group showed a lower in vitro cell survival rate (P < .001) compared with the other groups. Greater in vivo inhibition of xenograft tumor growth was also observed in the sunitinib-loaded microbubble + US group compared with the other groups. CONCLUSIONS: Combined sunitinib-loaded microbubbles and US treatment significantly inhibits growth of renal carcinoma cells both in vitro and in vivo.

[Clinical epidemiological study of peri-conceptional multiple risk factors and congenital heart diseases].

OBJECTIVE: To evaluate the associations between the occurrence of congenital heart diseases (CHDs) in children and exposures to peri-conceptional multiple nongenetic risk factors. METHODS: For this age-matched case-control study, standardized data were collected from 435 families of children with CHD and 574 families of non-malformed children. The questionnaires were conducted on multiple nongenetic risk factors including paternal characteristics and conditions, maternal therapeutic drug exposure, housing renovation, hair perming and dyeing and parental occupational exposure. RESULTS: The CHD infants weighed less than control group (P < 0.01). However two groups had no significant differences in age, height, parental age or heart rate. Educational levels of parents had a negative correlation with CHD. Conotruncal defect and simple ventricular septum defect were the most common cardiac abnormalities. Therapeutic drug exposure, viral infection and occupational exposure all had a high level of correlation with conotruncal defect (OR = 6.74, OR = 3.73, OR = 2.82 respectively). And ventricular septum defect was associated with therapeutic drug exposure, home decoration, hair perming and dyeing and parental exposure of work environment (OR = 2.77, OR = 1.76, OR = 1.89 respectively). CONCLUSION: Multiple nongenetic risk factors may contribute to the occurrence of CHD.

Ultrasound-guided percutaneous laser and ethanol ablation of rabbit VX2 liver tumors.

BACKGROUND: Only a small percentage of patients with hepatocellular carcinoma (HCC) may benefit out of surgical resection. Thus, lots of these patients are in need of local control, such as percutaneous ethanol injection (PEI), percutaneous laser ablation (PLA), or radiofrequency thermal ablation (RF). PURPOSE: To investigate the effects of ultrasound-guided PLA combined with PEI on rabbit VX2 liver tumors, using conventional gray-scale ultrasonography (US), color/power Doppler (CD/PD)US, contrast-enhanced (CE) US, and histologic examination. MATERIAL AND METHODS: VX2 tumors were implanted in the livers of 80 rabbits. Fourteen days after implantation, animals were randomly separated into four groups of 20 rabbits. Treatment of the four groups was with: (i) PLA; (ii) PEI; (iii) combined therapy of PLA immediately followed by PEI; and (iv) combined therapy of PEI immediately followed by PLA. Conventional gray-scale US, CD US, PD US, and CE US were performed before and after ablation. The effects on ablated areas were assessed by histologic examination. RESULTS: Conventional gray-scale US showed a clear boundary around the ablated area in groups 1, 3, and 4. An isoechoic treated region with an irregular boundary was seen in group 2. On CE US, coagulated areas demonstrated a perfusion defect. Both conventional gray-scale US and CE US showed that the ablated volume in group 4 was larger than that in groups 1, 2, and 3. CD US and PD US demonstrated residual tumor in the periphery of ablated areas in groups 1 and 2, but not in groups 3 and 4. CE US demonstrated no residual tumor in group 4, unlike in groups 1, 2, and 3. Examination of treated tumors demonstrated necrosis in the ablated zones and increasing surrounding fibrous bands in the four treatment groups. Residual viable tissue in group 4 was less than that in groups 1, 2, and 3. CONCLUSION: Combined therapy of PEI immediately followed by PLA can coagulate significantly larger volumes of tumor and reduce residual tumor.

Correlation between myocardial dysfunction and perfusion impairment in diabetic rats with velocity vector imaging and myocardial contrast echocardiography.

The purpose of this study was to investigate whether myocardial systolic dysfunction and perfusion impairment occur in diabetic rats, and to assess their relationship using velocity vector imaging (VVI) and myocardial contrast echocardiography (MCE). Forty-six rats were randomly divided into either control or the diabetes mellitus (DM) groups. DM was induced by intraperitoneal administration of streptozotocin. Twelve weeks later, 39 survival rats underwent VVI and MCE in short-axis view at the middle level of the left ventricle, both at rest and after dipyridamole stress. VVI-derived contractile parameters included peak systolic velocity (Vs ), circumferential strain (εc ), strain rate (SRc ), and their reserves. MCE-derived perfusion parameters consisted of myocardial blood flow (MBF) and myocardial flow reserve (MFR). At rest, SRc in the DM group was significantly lower than in the control group, Vs , εc , and MBF did not differ significantly between groups. After dipyridamole stress, all VVI parameters and their reserves in the DM group were significantly lower than those in the control group, MBF and MFR were substantially lower than those in the control group, too. Meanwhile, significant correlations between VVI parameter reserves and MFR were observed in the DM group. Both myocardial systolic function and perfusion were impaired in DM rats. Decreased MFR could be an important contributor to the reduction in myocardial contractile reserve.

A designed zinc-finger transcriptional repressor of phospholamban improves function of the failing heart.

Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.

Targeted ultrasound contrast imaging of matrix metalloproteinase-2 in ischemia-reperfusion rat model: ex vivo and in vivo studies.

PURPOSE: We hypothesized that post-myocardial ischemia-reperfusion (I/R) remodeling associated matrix metalloproteinase-2 (MMP(2)) activation could be detected by using novel MMP(2) targeted ultrasound imaging. PROCEDURES: We study the combination of MMP(2)-targeted microbubbles (TMB(2)) and control microbubbles with myocardium in 1 week post-I/R rats. RESULTS: In in vitro studies, TMB(2) significantly bound within the risk area (RA) of 1-week post-I/R myocardial sections while rare binding was observed in the control area (CA). In in vivo studies, increased focal retention of TMB(2) was observed within the RA, with the higher myocardial video intensity (RA 42.85 ± 20.12 dB versus CA 25.85 ± 13.40 dB, p < 0.01). However, there was no difference of control microbubble retention in both CA and RA. CONCLUSIONS: A targeted ultrasound contrast imaging approach that employs novel TMB(2) has the potential to provide a less-invasive, higher-resolution technique for in vivo localization of MMP(2) activation and tracking of MMP-mediated post-I/R remodeling.

Clinical application of contrast-enhanced ultrasonography in diagnosis of superficial lymphadenopathy.

OBJECTIVE: The purpose of this study was to evaluate the value of contrast-enhanced ultrasonography (CEUS) in differential diagnosis of superficial lymphadenopathy. METHODS: Ninety-four superficial enlarged lymph nodes in 94 patients were studied by conventional ultrasonography (gray scale and color Doppler) and CEUS. Contrast-enhanced sonograms were analyzed using contrast-specific quantification software. All of the results were compared with pathologic diagnoses. RESULTS: Of the 94 lymph nodes examined, 44 were benign and 50 were malignant (33 metastases and 17 lymphomas). The sensitivity, specificity, and accuracy of conventional ultrasonography in differential diagnosis between benign and malignant nodes were 51%, 47%, and 55%, respectively. Contrast-enhanced ultrasonography showed intense homogeneous enhancement in 39 of 44 benign lymph nodes, inhomogeneous enhancement in 32 of 33 metastases, and intense homogeneous enhancement and absence of perfusion in 9 of 17 and 6 of 17 lymphomas, respectively. The sensitivity specificity, and accuracy of CEUS were 84%, 79%, and 80%. After time-intensity curve gamma variates were calculated, the area under the curve of the benign lymph nodes was greater than those of the metastatic lymph nodes and lymphomas (P < .01). CONCLUSIONS: These results indicate that the use of CEUS and contrast-specific software has a higher degree of diagnostic accuracy than conventional ultrasonography for evaluations of superficial lymphadenopathy. The contrast enhancement patterns and time-intensity curves provide valuable diagnostic information for differential diagnosis of benign and malignant lymph nodes.

Effects of distal protection on left ventricular function in acute anterior myocardial infarction: a Doppler echocardiographic study.

Whether distal protection devices (DPDs) during percutaneous coronary intervention (PCI) can improve myocardial function in patients with acute myocardial infarction (AMI) is still under debate. Using tissue Doppler imaging (TDI), we evaluate the global and regional left ventricular systolic and diastolic functions in patients with anterior AMI using DPDs compared with conventional PCI. Ninety-six patients with anterior AMI were randomly assigned to either PCI with DPDs (DPD, n = 46) or traditional PCI (control, n = 50) groups. At the 3- and 6-month follow-ups, the DPD group had a higher left ventricular ejection fraction than the control group (51.6 +/- 3.6 vs. 49.3 +/- 5.3% and 53.0 +/- 3.7 vs. 50.8 +/- 5.2%, respectively; both P < 0.05). Moreover, peak systolic (S (a)) and early diastolic (E (a)) mitral annular velocities obtained by TDI were significantly higher in the DPD group than in the control group (S (a): 7.57 +/- 0.53 vs. 7.12 +/- 0.62 cm/s and 7.71 +/- 0.63 vs. 7.32 +/- 0.59 cm/s; E (a): 7.23 +/- 0.78 vs. 6.89 +/- 0.86 cm/s and 7.49 +/- 0.69 vs. 7.04 +/- 0.85 cm/s, respectively; all P < 0.05). However, systolic and diastolic regional myocardial velocities significantly improved in the DPD group from the 1-month follow-up compared with those in the control group (all P < 0.05). Patients who received treatment with DPDs experienced enhanced improvement of cardiac function. Thus, anterior AMI patients can benefit from DPDs during PCI.

Real-time myocardial contrast echocardiography in rat: infusion versus bolus administration.

To compare the feasibility of real-time myocardial contrast echocardiography (MCE) in rats with infusion and bolus administration of a second-generation ultrasound contrast agent BR1. B-mode real-time MCE was performed in 12 Sprague Dawley rats following the BR1 infusion or bolus injection. The myocardium signal intensity (SI) was plotted against time and was fitted to exponential functions. The plateau SI (A) and rate of SI increase (beta) for the infusion study and peak signal intensity (PSI) for the bolus study were obtained. (99m)Tc-Sestamibi and Evans blue were used to assess myocardial blood perfusion and to calculate the myocardium perfusion defect area ex vivo. High-quality real-time MCE images were successfully obtained using each method. At baseline, all LV segments showed even contrast distribution. Following left anterior descending coronary artery (LAD) ligation, significant perfusion defect was observed in LAD beds with a significantly decreased A* beta and PSI values compared with LCx beds (Infusion: A*beta (LAD): 5.42 +/- 1.57 dB, A*beta (LCx): 46.52 +/- 5.32 dB, p < 0.05; Bolus: PSI (LAD): 2.11 +/- 0.67 dB, PSI (LCx): 20.68 +/- 0.72 dB, p < 0.05), which was consistent with (99m)Tc-Sestamibi distribution findings. Myocardial perfusion defect areas, assessed by both methods, showed no differences and showed good correlation with Evans blue staining. ED frames were more favorable for imaging analysis. Both infusion and bolus administration of the contrast agent combined with real-time MCE technique can provide a reliable and noninvasive approach for myocardial perfusion assessment in rats and the infusion method was more suitable for quantitative analysis of myocardial blood flow.

Assessment of left ventricular wall motion in diabetic rats using velocity vector imaging combined with stress echocardiography.

OBJECTIVE: The aim of this study was to investigate whether velocity vector imaging (VVI) combined with stress echocardiography could detect potential diffused myocardial impairment of the left ventricle (LV) in diabetic rats. METHODS: Thirty-five male SD rats were randomly divided into either the control group or the diabetes mellitus (DM) group (induced with STZ). VVI was performed both at rest and after dipyridamole stress in all rats 12 weeks later. Segmental peak systolic velocity (V(s)), diastolic velocity (V(d)), radial strain (epsilon(r)), circumferential strain (epsilon(c)), systolic and diastolic radial strain rate (SR(r)), and circumferential strain rate (SR(c)) were measured from six segments at the mid-level of the LV. RESULTS: At rest, systolic and diastolic SR(c) in the DM group were significantly lower than those in the control group. After dipyridamole stress, all VVI parameters in the DM group were significantly lower than those in the control group, although all values increased significantly after dipyridamole stress compared to those at rest in both groups. CONCLUSIONS: The VVI-derived V(s,) V(d), epsilon(r), epsilon(c), systolic and diastolic SR(r) and SR(c), combined with dipyridamole stress are all effective parameters in evaluating potential myocardial impairment due to ultrastructural alterations of cardiocytes and microcirculation disturbances in DM rats. Systolic and diastolic SR(c) may be more sensitive indices that could be useful in detecting myocardial impairment at rest.

Hemostasis of active bleeding from the liver with percutaneous microwave coagulation therapy under contrast-enhanced ultrasonographic guidance: an experimental study.

OBJECTIVE: The purpose of this study was to investigate the feasibility of percutaneous microwave coagulation therapy (PMCT) guided by contrast-enhanced ultrasonography (CEUS) for controlling active bleeding in rabbit livers. METHODS: Twenty actively bleeding rabbit liver models, produced with an 18-gauge semiautomatic biopsy needle and confirmed with CEUS, were randomly divided into 2 groups: a PMCT group (n=10, with a microwave antenna placed into the bleeding site under ultra-sonographic guidance and worked at 60 W for 30 seconds on average) and a control group (n=10, with the active bleeding site not treated). After therapy procedures were performed, lactated Ringer's solution resuscitation was then performed in both groups to maintain the mean arterial pressure at 70 mm Hg for 1 hour. The intraperitoneal blood loss, total resuscitation volume, mean arterial pressure, and hematocrit value were recorded. Macroscopic and microscopic examinations were performed at the end of the study. RESULTS: After PMCT, the former bleeding site appeared on CEUS as a round or an oval area devoid of contrast. The PMCT group had lower blood loss (30.4+/-7.2 versus 101.6 +/- 18.2 mL; P< .05) and a lower total resuscitation volume (56.5+/-10 versus 186+/-36.6 mL; P< .05) than the control group. The mean hematocrit value in the PMCT group was significantly higher than that in the control group (26%+/-4% versus 19%+/-4%; P< .05) at the end of the experiment. CONCLUSIONS: Contrast-enhanced ultrasonographically guided PMCT significantly decreased blood loss in a rabbit model of active liver bleeding. It provides a simple and quick method to control blood loss in liver injuries with active bleeding.

Development of a model system for preliminary evaluation of tissue-engineered vascular conduits.

BACKGROUND/PURPOSE: The ability to construct tissue-engineered neovessels for use as arterial or venous grafts holds great promise for the advancement of pediatric surgical disciplines. Although the feasibility of tissue engineering vascular grafts has been demonstrated, the long-term function, safety, and efficacy of these grafts as well as their capacity to grow and adapt remain largely unknown. In an attempt to further characterize and develop this technology, we used severe combined immunodeficiency beige (SCID/bg) mouse recipients, chosen because such animals accept xenogenic human cells, to create a small animal model that would allow a rapid and cost-effective preliminary evaluation of the function of tissue-engineered vascular grafts. METHODS: Eight CB-17 SCID/bg female mice underwent vascular graft placement. Four of these mice received aortic interposition grafts, 1 mouse received an inferior vena cava interposition graft, and 3 mice underwent aortocaval graft insertion. All grafts were fashioned from decellularized ovine arteriole tissue engineering scaffolds. Grafts were evaluated for patency using clinical examination, ultrasound interrogation, and micro-computed tomography. Animals were killed at various time points after implantation, and grafts were harvested and analyzed histologically using standard hematoxylin and eosin staining. RESULTS: All grafts were patent based on clinical examination for up to 35 days. Patency was confirmed in 5 grafts using ultrasound interrogation. Patency was confirmed in 4 grafts using micro-computed tomography. One animal that underwent arteriovenous grafting had to be euthanized secondary to high-output cardiac failure on postoperative day 2. The remaining animals were killed between postoperative days 12 and 35. Histologic evaluation of the specimens demonstrated patent grafts with cellular ingrowth into the tissue engineering scaffold. CONCLUSIONS: From these results, we conclude that the use of the SCID/bg mouse model for preliminary evaluation of new tissue engineering methodologies for construction of vascular conduits is feasible. Use of this model has the added advantage of evaluating nonautologous and even xenograft tissues, including human cells.

Matrix metalloproteinase-9 gene deletion facilitates angiogenesis after myocardial infarction.

Matrix metalloproteinases (MMPs) are postulated to be necessary for neovascularization during wound healing. MMP-9 deletion alters remodeling postmyocardial infarction (post-MI), but whether and to what degree MMP-9 affects neovascularization post-MI is unknown. Neovascularization was evaluated in wild-type (WT; n = 63) and MMP-9 null (n = 55) mice at 7-days post-MI. Despite similar infarct sizes, MMP-9 deletion improved left ventricular function as evaluated by hemodynamic analysis. Blood vessel quantity and quality were evaluated by three independent studies. First, vessel density was increased in the infarct of MMP-9 null mice compared with WT, as quantified by Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I) immunohistochemistry. Second, preexisting vessels, stained in vivo with FITC-labeled GSL-I pre-MI, were present in the viable but not MI region. Third, a technetium-99m-labeled peptide (NC100692), which selectively binds to activated alpha(v)beta3-integrin in angiogenic vessels, was injected into post-MI mice. Relative NC100692 activity in myocardial segments with diminished perfusion (0-40% nonischemic) was higher in MMP-9 null than in WT mice (383 +/- 162% vs. 250 +/- 118%, respectively; P = 0.002). The unique finding of this study was that MMP-9 deletion stimulated, rather than impaired, neovascularization in remodeling myocardium. Thus targeted strategies to inhibit MMP-9 early post-MI will likely not impair the angiogenic response.

Noninvasive targeted imaging of matrix metalloproteinase activation in a murine model of postinfarction remodeling.

BACKGROUND: Time-dependent activation of matrix metalloproteinases (MMPs) after myocardial infarction (MI) contributes to adverse left ventricular (LV) remodeling; however, noninvasive methods to monitor this process serially are needed. METHODS AND RESULTS: MMP-targeted radiotracers were developed that displayed selective binding kinetics to the active MMP catalytic domain. Initial nonimaging studies were performed with a (111)In-labeled MMP-targeted radiotracer ((111)In-RP782) and negative control compound ((111)In-RP788) in control mice (Ctrl) and in mice 1 week after surgically induced MI. Localization of (111)In-RP782 was demonstrated within the MI by microautoradiography. A 334+/-44% increase (P<0.001 versus Ctrl) in relative retention of (111)In-RP782 was confirmed by gamma well counting of myocardium. Subsequent high-resolution dual-isotope planar and hybrid micro-single-photon emission computed tomography/CT imaging studies with an analogous 99mTc-labeled MMP-targeted radiotracer (99mTc-RP805) and 201Tl demonstrated favorable biodistribution and clearance kinetics of 99mTc-RP805 for in vivo cardiac imaging, with robust retention 1 to 3 weeks after MI in regions of decreased 201Tl perfusion. Gamma well counting yielded a similar approximately 300% increase in relative myocardial retention of 99mTc-RP805 in MI regions (Ctrl, 102+/-9%; 1 week, 351+/-77%; 2 weeks, 291+/-45%; 3 weeks, 292+/-41%; P<0.05 versus Ctrl). Myocardial uptake in the MI region was also significantly increased approximately 5-fold when expressed as percentage injected dose per gram tissue. There was also a significant 2-fold increase in myocardial activity in remote regions relative to control mice, suggesting activation of MMPs in regions remote from the MI. CONCLUSIONS: This novel noninvasive targeted MMP radiotracer imaging approach holds significant diagnostic potential for in vivo localization of MMP activation and tracking of MMP-mediated post-MI remodeling.

Noninvasive imaging of myocardial angiogenesis following experimental myocardial infarction.

Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The alphavbeta3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111-labeled ((111)In-labeled) alphavbeta3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted alphavbeta3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled alphavbeta3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission-computed tomographic (SPECT) imaging with the (111)In-labeled alphavbeta3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the alphavbeta3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an (111)In-labeled alphavbeta3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.

Intracellular localization of differentially regulated RNA-specific adenosine deaminase isoforms in inflammation.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation.