Biomedical subjects

Lawrence W Dobrucki

Publications and source records attributed to Lawrence W Dobrucki.

15 recordsLinked to original sources

PubMed2026

Cu(II/I)-Pyridinophane Complexes: O2 Reactivity and 64Cu PET Imaging Studies.

The redox versatility of copper between the Cu(I) and Cu(II) oxidation states is central to its function in a variety of biological processes, including electron transfer, oxygen transport, and redox catalysis by metalloenzymes. Motivated by these biological paradigms, we report the design and development of a new series of chelators based on the 2-aza[3,2](2,6)pyridinophane (RN3) ligands (RN3, R = H, Me, 2-methylpyridyl, and 2-methylpicolinate). These ligands are designed to replicate key features of metalloenzyme active sites and to modulate the copper coordination environment. The new RN3 ligands have been fully characterized by NMR spectroscopy, high-resolution mass spectrometry, and UV-vis spectrophotometric titrations. The Cu(I) and Cu(II) complexes supported by these ligands were synthesized and characterized structurally and spectroscopically to investigate the effects of ligand denticity, variation in donor atoms, and chelate ring size on coordination geometry, bond parameters, and redox behavior. Special attention is given to the reactivity of Cu(I) complexes toward O2, offering insights into pathways of O2 activation and reduction. The RN3 ligands also demonstrate promising stability and binding affinity for both Cu(II) and Cu(I) ions, underscoring their potential for 64Cu-based positron emission tomography (PET) imaging applications.

Journal Article↗

PubMed2006

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

Noninvasive imaging of angiogenesis with a 99mTc-labeled peptide targeted at alphavbeta3 integrin after murine hindlimb ischemia.

BACKGROUND: Noninvasive imaging strategies play a critical role in assessment of the efficacy of angiogenesis therapies. The alpha(v)beta3 integrin is activated in angiogenic vessels and represents a potential target for noninvasive imaging of angiogenesis. METHODS AND RESULTS: We evaluated a 99mTc-labeled peptide (NC100692) targeted at alpha(v)beta3 integrin for imaging in an established murine model of angiogenesis induced by hindlimb ischemia. Control mice (n=9) or mice with surgical right femoral artery occlusion (n=29) were injected with NC100692 (1.5+/-0.2 mCi IV) at different times after femoral occlusion (1, 3, 7, and 14 days) for in vivo pinhole planar gamma camera imaging. Tissue from hindlimb proximal and distal to occlusion was excised for gamma well counting and for immunostaining. On in vivo pinhole images, increased focal NC100692 activity was seen distal to the occlusion at days 3 and 7. This increase in relative NC100692 activity was confirmed by gamma well counting. Lectin staining confirmed increased angiogenesis in the ischemic hindlimb at these time points. A fluorescent analogue of NC100692 was used to confirm specificity and localization of the targeted tracer in cultured endothelial cells. In addition, endothelial cell specificity was confirmed on tissue sections with the use of dual immunofluorescent staining of endothelium and the fluorescent analogue targeted at the alpha(v)beta3 integrin. CONCLUSIONS: A 99mTc-labeled peptide (NC100692) targeted at alpha(v)beta3 integrin selectively localized to endothelial cells in regions of increased angiogenesis and could be used for noninvasive serial "hot spot" imaging of angiogenesis. This targeted radiotracer imaging approach is a major advance in tracking therapeutic myocardial angiogenesis and has an important clinical potential.

Animals↗

PubMed2005

Articulated rigid registration for serial lower-limb mouse imaging.

This paper describes a new piecewise rotational transformation model for capturing the articulation of joints such as the hip and the knee. While a simple piecewise rigid model can be applied, such models suffer from discontinuities at the motion boundary leading to both folding and stretching. Our model avoids both of these problems by constructing a provably continuous transformation along the motion interface. We embed this transformation model within the robust point matching framework and demonstrate its successful application to both synthetic data, and to serial x-ray CT mouse images. In the later case, our model captures the articulation of six joints, namely the left/right hip, the left/right knee and the left/right ankle. In the future such a model could be used to initialize non-rigid registrations of images from different subjects, as well as, be embedded in intensity-based and integrated registration algorithms. It could also be applied to human data in cases where articulated motion is an issue (e.g. image guided prostate radiotherapy, lower extremity CT angiography).

Algorithms↗

PubMed2005

A new method for SPECT quantification of targeted radiotracers uptake in the myocardium.

We developed a new method for absolute quantification of targeted radiotracers uptake in the myocardium using hybrid SPECT/CT and an external reference point source. A segmentation algorithm based on the level set was developed to determine the endocardial edges from CT, which were subsequently applied to the physically co-registered SPECT. A 3-D Gaussian fitting method was applied for quantification of the external point source. The total targeted radiotracer activity in the myocardium was normalized to that in the point source to calculate the absolute uptake of targeted radiotracer in the myocardium. Preliminary validation was performed in rats with ischemia-induced angiogenesis. The quantified in vivo radiotracer uptake was compared to the postmortem tissue radioactive well-counting of the myocardium. Our methods worked well for identification of the endocardial edges. Quantification of the focal uptake was consistent with the well-counting data. Our methods may have the potential of providing precise absolute quantification of targeted radiotracer uptake in the myocardium.

Animals↗

PubMed2005

Characterizing vascular connectivity from microCT images.

X-ray microCT (computed tomography) has become a valuable tool in the analysis of vascular architecture in small animals. Because of its high resolution, a detailed assessment of blood vessel physiology and pathology is possible. Vascular measurement from noninvasive imaging is important for the study and quantification of vessel disease and can aid in diagnosis, as well as measure disease progression and response to therapy. The analysis of tracked vessel trajectories enables the derivation of vessel connectivity information, lengths between vessel junctions as well as level of ramification, contributing to a quantitative analysis of vessel architecture. In this paper, we introduce a new vessel tracking methodology based on wave propagation in oriented domains. Vessel orientation and vessel likelihood are estimated based on an eigenanalysis of gray-level Hessian matrices computed at multiple scales. An anisotropic wavefront then propagates through this vector field with a speed modulated by the maximum vesselness response at each location. Putative vessel trajectories can be found by tracing the characteristics of the propagation solution between different points. We present preliminary results from both synthetic and mouse microCT image data.

Algorithms↗

PubMed2005

Molecular cardiovascular imaging.

Imaging with radionuclides has historically played an important role in detection of cardiovascular disease as well as in risk stratification and prognostication. With the growth of molecular biology have come new therapeutic interventions and the requirement for new diagnostic imaging approaches. Noninvasive targeted radiotracer-based strategies require the development of new instrumentation to meet these needs. This progress has been made possible with the availability of many technologic advances, which include dedicated micro single-photon emission computed tomography (SPECT) and micro positron emission tomography (PET) hybrid systems for small animal imaging. This review is a brief overview on the subject of molecular imaging. Basic concepts of molecular imaging are reviewed, followed by description of current technologic advances, and current applications to evaluate ischemic heart disease and potential therapeutic intervention. The emphasis is on the use of both SPECT and PET radiotracers, although other imaging modalities are also briefly discussed.

Angiogenesis Inducing Agents↗

PubMed2005

Cardiovascular molecular imaging.

The recent introduction of novel gene therapies for treatment of cardiac and noncardiac diseases has caused a remarkable need for noninvasive imaging approaches to evaluate and track the progress of these therapies. In the past we have relied on the evaluation of the physiological consequences of therapeutic interventions. With advances in targeted molecular imaging we now have the ability to evaluate early molecular effects of these therapies. The development of dedicated high resolution small animal imaging systems and the establishment of transgenic animal models has enhanced our understanding of cardiovascular disease and has expedited the development of new gene therapies. Noninvasive targeted molecular imaging will allow us to directly track biochemical processes and signaling events that precede the pathophysiological changes. The examples of targeted molecular imaging outlined in this seminar provide some insight into the bright and growing future of cardiovascular molecular imaging. The success of this new field rests on the development of targeted biological markers of molecular and physiological processes, development of new instruments with improved sensitivity and resolution, and the establishment of multidisciplinary teams of experimental and clinical investigators with a wide range of expertise. Molecular imaging already plays a critical role in the experimental laboratory. We expect that, in the near future, targeted molecular imaging will be routinely used in clinical cardiovascular nuclear medicine laboratories in conjunction with existing imaging modalities for both diagnostic and prognostic purposes, as well as for evaluation of new genetic based therapeutic strategies.

Animals↗

PubMed2004

Combined L-arginine and antioxidative vitamin treatment mollifies ischemia-reperfusion injury of skeletal muscle.

Enhanced production of superoxide in L-arginine-depleted environments and concomitant reduction of nitric oxide (NO) concentration are involved in ischemia-reperfusion (I/R) injury. Treatment with L-arginine or antioxidative vitamins alone and in combination was used to mollify I/R injury in skeletal muscle. Untreated rabbits were compared with those treated with L-arginine/antioxidative vitamin cocktail Omnibionta only, or a combination of L-arginine/ antioxidative vitamins during hind limb I/R (2.5 hours/2 hours). NO was continuously measured in vivo. Plasma malondialdehyde (MDA) served as the measure of oxygen free radical formation. Interstitial edema formation, microvessel diameter alterations, microvessel plugging, and blood flow changes were used as indicators of I/R injury. The MDA level in untreated animals 2 hours after reperfusion was significantly higher than in control animals (0.81 micromol/L +/- 0.14 micromol/L vs 0.57 micromol/L +/- 0.11 micromol/L; P<.05), indicating enhanced production of oxygen free radicals. This sequela paralleled the decreasing concentration of NO, which dropped below the detection limit (1 nmol/L) after reperfusion. Microvascular changes during I/R injury were expressed as a 40% decrease in microvessel diameter and adhesion of neutrophils in 20% of microvessels, which led to a consequent 60% reduction in blood flow, demonstrating "no reflow" (reperfusion failure after restoration of blood flow). The increase in the fraction of muscle interfiber area by 85% indicated prominent edema formation. Treatment with antioxidative vitamins alone had a minimally positive effect on edema formation and microvascular plugging, possibly by suppression of oxygen free radical production, as expressed by the reduction in plasma MDA levels. However, this therapy failed to preserve basal NO production and to protect from microvascular constriction and no reflow. Treatment with L-arginine alone had a stronger protective effect, maintaining basal NO production, further reduction of neutrophil plugging, abolition of microvascular constriction, and no reflow. The combination of antioxidative vitamins and L-arginine was the best treatment against I/R injury, expressed not only by the protection of microvessel constriction, but also by abolition of microvascular plugging, increase in NO production (68 nmol/L +/- 5 nmol/L) over the basal level (52 nmol/L +/- 7 nmol/L), and higher blood flow, as compared with treatment with L-arginine or antioxidative vitamins alone.

Amino Acids↗

PubMed2003

Third-generation beta-blockers stimulate nitric oxide release from endothelial cells through ATP efflux: a novel mechanism for antihypertensive action.

BACKGROUND: Nebivolol and carvedilol are third-generation beta-adrenoreceptor antagonists, which unlike classic beta-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. METHODS AND RESULTS: Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [3H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both beta-blocker-induced glomerular vasorelaxations and beta-blocker-stimulated NO release from GECs. Both beta-blocker-induced vasorelaxations were in the micromolar concentration range identical to that required for the beta-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188+/-14 and 226+/-17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd3+, inhibited the beta-blocker-dependent release of ATP and NO from GECs. CONCLUSIONS: These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor-mediated NO release from GECs.

Adenosine Triphosphate↗

PubMed2003

Ramipril improves nitric oxide availability in hypertensive rats with failing hearts after myocardial infarction.

A markedly decreased aortic nitric oxide (NO) availability, probably due to impaired endothelial nitric oxide synthase activity with enhanced O2- and peroxynitrite production, seems to be attributable to endothelial dysfunction in spontaneously hypertensive rats (SHR) with severe congestive heart failure (CHF). In this study, we investigated the chronic effect of the angiotensin-converting enzyme inhibitor, ramipril (RA) and the loop diuretic, frusemide (FU), as well as the combination of both on endothelial NO, O2- and peroxynitrite production in aortae from SHR with failing hearts after myocardial infarction (MI). Heart failure was induced by permanent occlusion of the left coronary artery. SHR were randomised to receive either placebo, RA, (1 mg/kg/day), FU (4 mg/kg/day) or RA+FU (1 and 4 mg/kg/day, respectively). Treatments were started two weeks following MI and continued for six weeks. Reduced aortic and coronary flow indices in the working heart, which can be considered as markers for endothelial function, were significantly normalised and improved, respectively, by RA, FU or RA+FU-treatment. Similarly, all three treatment regimens significantly enhanced the reduced calcium ionophore (CaI)-induced NO-release (assessed by a NO-sensitive microsensor) from aortic endothelial cells of placebo-treated animals with CHF. Concomitantly, the increased CaI-stimulated O2- production (assessed by an electrochemical sensor) in aortic endothelial cells of placebo-treated animals with CHF was significantly reduced by RA and RA+FU-treatment. Treatment with RA and RA+FU also attenuated the dramatic increase in endothelial peroxynitrite concentration (chemiluminescence method), which was observed in placebo-treated rats with CHF. FU did not counteract improved haemo- and cardiodynamic parameters by RA. Thus, RA and FU act synergistically to enhance bioavailability of endothelium-derived NO, and this may contribute to the clinical usefulness of the combination of these drugs in treatment of heart failure.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2002

Increased nitric oxide bioavailability in endothelial cells contributes to the pleiotropic effect of cerivastatin.

BACKGROUND: Although statins preserve endothelial function by reducing serum cholesterol levels, it has been suggested they may also stimulate nitric oxide (NO) synthase in endothelium with concurrent increase in superoxide (O2-) generation, leading to impairment of NO activity. Therefore, measurements of biologically active NO and O2- in endothelium after exposure to the HMG-CoA reductase inhibitor cerivastatin were undertaken to evaluate its potential effect on NO biological activity. METHODS AND RESULTS: Highly sensitive electrochemical NO and O2- microsensors were placed near the surface of a single human umbilical vein endothelial cell, and the kinetics of NO and O2- release were recorded in vitro. Cerivastatin demonstrated a time-dependent effect on NO release in endothelial cells. The initial release (approximately the first 3 minutes) was concentration-dependent (0.01 to 10 micromol/L) and was similar to that observed for typical NO synthase agonists calcium ionophore or acetylcholine. Cerivastatin stimulated NO release at a favorable rate and scavenged O2-, which led to the preservation of the active concentration of NO. The sustained effect (after approximately 6 hours) of cerivastatin on endothelium was associated with an approximately 35% increase in NO release as compared with the initial effect. In contrast to the initial effect, the sustained effect of cerivastatin was shown at concentrations approximately 100-fold lower and was dependent on inhibition of endothelial HMG-CoA reductase. CONCLUSIONS: These data provide direct evidence to prove that in the presence of cerivastatin, the NOS system in endothelium operates with high efficiency toward increasing NO activity by activation of NO release and by concurrent inactivation of O2-.

Biological Availability↗

PubMed2002

AVE 0991, a nonpeptide mimic of the effects of angiotensin-(1-7) on the endothelium.

Recently, we demonstrated that the heptapeptide angiotensin-(1-7) (Ang-[1-7]) exhibits a favorable kinetic of nitric oxide (NO) release accompanied by extremely low superoxide (O2-) production. In this report we describe AVE 0991, a novel nonpeptide compound that evoked effects similar to Ang-(1-7) on the endothelium. AVE 0991 and unlabeled Ang-(1-7) competed for high-affinity binding of [125I]-Ang-(1-7) to bovine aortic endothelial cell membranes with IC50 values of 21+/-35 and 220+/-280 nmol/L, respectively. Stimulated NO and O2- release from bovine aortic endothelial cells was directly and simultaneously measured on the cell surface by selective electrochemical nanosensors. Peak concentrations of NO and O2- release by AVE 0991 and Ang-(1-7) (both 10 micromol/L) were not significantly different (NO: 295+/-20 and 270+/-25 nmol/L; O2-: 18+/-2 and 20+/-4 nmol/L). However, the released amount of bioactive NO was approximately 5 times higher for AVE 0991 in comparison to Ang-(1-7). The selective Ang-(1-7) antagonist [D-Ala(7)]-Ang-(1-7) inhibited the AVE 0991-induced NO and O2- production by approximately 50%. A similar inhibition level was observed for the Ang II AT1 receptor antagonist EXP 3174. In contrast, the Ang II AT2 receptor antagonist PD 123,177 inhibited the AVE 0991-stimulated NO production by approximately 90% but without any inhibitory effect on O2- production. Both NO and O2- production were inhibited by NO synthase inhibition ( approximately 70%) and by bradykinin B2 receptor blockade (approximately 80%). AVE 0991 efficiently mimics the effects of Ang-(1-7) on the endothelium, most probably through stimulation of a specific, endothelial Ang-(1-7)-sensitive binding site causing kinin-mediated activation of endothelial NO synthase.

Angiotensin I↗