[Show abstract][Hide abstract] ABSTRACT: Detection of breast cancer by positron emission tomography (PET) imaging with 2-(fluorine-18)-2-deoxy-d-glucose (FDG) as the tracer molecule is limited in part by both tumor dimension and metabolic activity. While some types of aggressive breast cancers are associated with a high capacity for FDG uptake, more indolent breast cancers are characterized by low FDG uptake. Moreover, detection of malignant lesions in most clinical settings requires tumor dimensions ≥10 mm. Development of a method to increase the fractional uptake of FDG by cancer tissue would provide a means to detect smaller tumors. However, there is no clinically available pharmacologic reagent known to enhance the preferential uptake of FDG by cancer tissue. Because the vanadyl (VO(2+)) chelate bis(acetylacetonato)oxovanadium(iv) [VO(acac)2] is known to enhance cellular uptake of glucose, we have investigated whether VO(acac)2 facilitates enhanced uptake of FDG by cultured human breast carcinoma cells. We observed that the fractional uptake of FDG by cultured human MDA-MB-231 carcinoma cells is increased in the presence of VO(acac)2 in a dose dependent manner. Preliminary results with xenograft tumors generated in severely compromised, immunodeficient (SCID) female mice showed that VO(acac)2 treatment of mice 3-4 h prior to FDG injection enhanced FDG uptake by the malignant tissue by a factor >2.0 compared with that by normal surrounding tissue.
[Show abstract][Hide abstract] ABSTRACT: Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin-2 (Mfn-2), which promotes fusion, and dynamin-related protein (Drp-1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp-1/Mfn-2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp-1 and decrease in Mfn-2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn-2, Drp-1 inhibition, or Drp-1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp-1 inhibition (P<0.001). In a xenotransplantation model, Mfn-2 gene therapy or Drp-1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm(3) (P<0.05) with Mfn-2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm(3) (P<0.01) with therapeutic Drp-1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.
The FASEB Journal 02/2012; 26(5):2175-86. DOI:10.1096/fj.11-196543 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pulmonary arterial hypertension (PAH) is a proliferative arteriopathy associated with glucose transporter-1 (Glut1) up-regulation and a glycolytic shift in lung metabolism. Glycolytic metabolism can be detected with the positron emission tomography (PET) tracer (18)F-fluorodeoxyglucose (FDG).
The precise cell type in which glycolytic abnormalities occur in PAH is unknown. Moreover, whether FDG-PET is sufficiently sensitive to monitor PAH progression and detect therapeutic regression is untested. We hypothesized that increased lung FDG-PET reflects enhanced glycolysis in vascular cells and is reversible in response to effective therapies.
PAH was induced in Sprague-Dawley rats by monocrotaline or chronic hypoxia (10% oxygen) in combination with Sugen 5416. Monocrotaline rats were treated with oral dichloroacetate or daily imatinib injections. FDG-PET scans and pulmonary artery acceleration times were obtained weekly. The origin of the PET signal was assessed by laser capture microdissection of airway versus vascular tissue. Metabolism was measured in pulmonary artery smooth muscle cell (PASMC) cultures, using a Seahorse extracellular flux analyzer.
Lung FDG increases 1-2 weeks after monocrotaline (when PAH is mild) and is normalized by dichloroacetate and imatinib, which both also regress medial hypertrophy. Glut1 mRNA is up-regulated in both endothelium and PASMCs, but not airway cells or macrophages. PASMCs from monocrotaline rats are hyperproliferative and display normoxic activation of hypoxia-inducible factor-1α (HIF-1α), which underlies their glycolytic phenotype.
HIF-1α-mediated Glut1 up-regulation in proliferating vascular cells in PAH accounts for increased lung FDG-PET uptake. FDG-PET is sensitive to mild PAH and can monitor therapeutic changes in the vasculature.
American Journal of Respiratory and Critical Care Medicine 01/2012; 185(6):670-9. DOI:10.1164/rccm.201108-1562OC · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.
[Show abstract][Hide abstract] ABSTRACT: Craniosynostosis is a significant disorder affecting 1 in 2500 live births worldwide. Although a large body of work has focused on dural regulation and the contributions of molecular mediators such as fibroblast growth factor, bone morphogenetic protein, and transforming growth factor β, minimal attention has been directed toward osteoclast function in cranial suture biology. Receptor activator of nuclear factor κB (RANK) is an essential mediator of osteoclastogenesis and osteoclast activation. In this study, physiologic fusion of posterior frontal sutures in murine development correlated with decreasing protein expression of RANK in comparison to age-matched coronal and sagittal sutures via immunohistochemical survey. However, RANK mRNA did not exhibit a similar pattern suggesting that RANK is regulated at the protein level. Fused cranial sutures in nonsyndromic craniosynostotic children also showed decreased levels of RANK staining in immunohistochemistry in comparison to patent sutures from the same patients. Immunohistochemistry with a RANK ligand antibody did not show differences in fused or patent sutures. Moreover, RANK knockdown in calvarial strip suture cultures displayed increased bone density specifically in the suture line after infection with small interfering RANK viruses. Cranial suture biology, similar to bone biology in general, likely depends on a complex interplay between osteoblasts and osteoclasts. We now report a temporospatial correlation between RANK expression and suture morphology that suggests that osteoclast activity is important in maintenance of cranial suture patency in normal physiology and disease. Furthermore, RANK downregulation promoted suture fusion establishing a causal relationship between the presence of RANK and patency.
The Journal of craniofacial surgery 03/2011; 22(2):699-705. DOI:10.1097/SCS.0b013e3182077fbd · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The etiology of hepatopulmonary syndrome (HPS), a common complication of cirrhosis, is unknown. Inflammation and macrophage accumulation occur in HPS; however, their importance is unclear. Common bile duct ligation (CBDL) creates an accepted model of HPS, allowing us to investigate the cause of HPS.
We hypothesized that macrophages are central to HPS and investigated the therapeutic potential of macrophage depletion.
Hemodynamics, alveolar-arterial gradient, vascular reactivity, and histology were assessed in CBDL versus sham rats (n = 21 per group). The effects of plasma on smooth muscle cell proliferation and endothelial tube formation were measured. Macrophage depletion was used to prevent (gadolinium) or regress (clodronate) HPS. CD68(+) macrophages and capillary density were measured in the lungs of patients with cirrhosis versus control patients (n = 10 per group).
CBDL increased cardiac output and alveolar-arterial gradient by causing capillary dilatation and arteriovenous malformations. Activated CD68(+)macrophages (nuclear factor-κB+) accumulated in HPS pulmonary arteries, drawn by elevated levels of plasma endotoxin and lung monocyte chemoattractant protein-1. These macrophages expressed inducible nitric oxide synthase, vascular endothelial growth factor, and platelet-derived growth factor. HPS plasma increased endothelial tube formation and pulmonary artery smooth muscle cell proliferation. Macrophage depletion prevented and reversed the histological and hemodynamic features of HPS. CBDL lungs demonstrated increased medial thickness and obstruction of small pulmonary arteries. Nitric oxide synthase inhibition unmasked exaggerated pulmonary vasoconstrictor responses in HPS. Patients with cirrhosis had increased pulmonary intravascular macrophage accumulation and capillary density.
HPS results from intravascular accumulation of CD68(+)macrophages. An occult proliferative vasculopathy may explain the occasional transition to portopulmonary hypertension. Macrophage depletion may have therapeutic potential in HPS.
American Journal of Respiratory and Critical Care Medicine 12/2010; 183(8):1080-91. DOI:10.1164/rccm.201008-1303OC · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate the feasibility of using a 2-deoxy-d-glucose (2-DG) labeled gold nanoparticle (AuNP-2-DG) as a functionally targeted computed tomography (CT) contrast agent to obtain high-resolution metabolic and anatomic information of tumor in a single CT scan. Gold nanoparticles (AuNPs) were fabricated and were conjugated with 1-DG or 2-DG. 1-DG provides an excellent comparison since it is known to interfere with the ability of the glucose transporter to recognize the sugar moiety. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Three groups of cell samples were incubated with the 1-DG or 2-DG labeled AuNP and the unlabeled AuNP. Following the incubation, the cells were washed with sterile phosphate buffered saline to remove the excess AuNPs and spun using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. Internalization of AuNP-2-DG is verified using transmission electron microscopy imaging. Significant contrast enhancement in the cell samples incubated with the AuNP-2-DG with respect to the cell samples incubated with the unlabeled AuNP and the AuNP-1-DG was observed in multiple CT slices. Results from our in vitro experiments suggest that the AuNP-2-DG may be used as a functional CT contrast agent to provide high-resolution metabolic and anatomic information in a single CT scan. These results justify further in vitro and in vivo experiments to study the feasibility of using the AuNP-2-DG as a functional CT contrast agent in radiation therapy settings.
Physics in Medicine and Biology 08/2010; 55(15):4389-97. DOI:10.1088/0031-9155/55/15/013 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP-9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin-like growth factor 2 (IGF-2) on BMP-9-induced bone formation. We have found that endogenous IGF-2 expression is low in MSCs. Expression of IGF-2 can potentiate BMP-9-induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF-2 has been shown to augment BMP-9-induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF-2 enhances BMP-9-induced endochondral ossification, whereas IGF-2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF-2 effect on BMP-9-induced ALP activity and matrix mineralization. Mechanistically, IGF-2 is further shown to enhance the BMP-9-induced BMPR-Smad reporter activity and Smad1/5/8 nuclear translocation. PI3-kinase (PI3K) inhibitor LY294002 abolishes the IGF-2 potentiation effect on BMP-9-mediated osteogenic signaling and can directly inhibit BMP-9 activity. These results demonstrate that BMP-9 crosstalks with IGF-2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP-9 and IGF-2 may be explored as an effective bone-regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 05/2010; 25(11):2447-59. DOI:10.1002/jbmr.133 · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the feasibility of using 2-deoxy-D-glucose (2-DG)-labeled gold nanoparticle (AuNP-DG) as a computed tomography (CT) contrast agent with tumor targeting capability through in vitro experiments.
Gold nanoparticles (AuNP) were fabricated and were conjugated with 2-deoxy-D-glucose. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Two groups of cell samples were incubated with the AuNP-DG and the unlabeled AuNP, respectively. Following the incubation, the cells were washed with sterile PBS to remove the excess gold nanoparticles and spun to cell pellets using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. The reconstructed CT images were analyzed using a commercial software package.
Significant contrast enhancement in the cell samples incubated with the AuNP-DG with respect to the cell samples incubated with the unlabeled AuNP was observed in multiple CT slices.
Results from this study demonstrate enhanced uptake of 2-DG-labeled gold nanoparticle by cancer cells in vitro and warrant further experiments to study the exact molecular mechanism by which the AuNP-DG is internalized and retained in the tumor cells.
Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 03/2010; 12(5):463-7. DOI:10.1007/s11307-010-0299-8 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Right ventricular hypertrophy (RVH) and RV failure contribute to morbidity and mortality in pulmonary arterial hypertension (PAH). The cause of RV dysfunction and the feasibility of therapeutically targeting the RV are uncertain. We hypothesized that RV dysfunction and electrical remodeling in RVH result, in part, from a glycolytic shift in the myocyte, caused by activation of pyruvate dehydrogenase kinase (PDK). We studied two complementary rat models: RVH + PAH (induced by monocrotaline) and RVH + without PAH (induced by pulmonary artery banding (PAB)). Monocrotaline RVH reduced RV O(2)-consumption and enhanced glycolysis. RV 2-fluoro-2-deoxy-glucose uptake, Glut-1 expression, and pyruvate dehydrogenase phosphorylation increased in monocrotaline RVH. The RV monophasic action potential duration and QT(c) interval were prolonged due to decreased expression of repolarizing voltage-gated K(+) channels (Kv1.5, Kv4.2). In the RV working heart model, the PDK inhibitor, dichloroacetate, acutely increased glucose oxidation and cardiac work in monocrotaline RVH. Chronic dichloroacetate therapy improved RV repolarization and RV function in vivo and in the RV Langendorff model. In PAB-induced RVH, a similar reduction in cardiac output and glycolytic shift occurred and it too improved with dichloroacetate. In PAB-RVH, the benefit of dichloroacetate on cardiac output was approximately 1/3 that in monocrotaline RVH. The larger effects in monocrotaline RVH likely reflect dichloroacetate's dual metabolic benefits in that model: regression of vascular disease and direct effects on the RV. Reduction in RV function and electrical remodeling in two models of RVH relevant to human disease (PAH and pulmonic stenosis) result, in part, from a PDK-mediated glycolytic shift in the RV. PDK inhibition partially restores RV function and regresses RVH by restoring RV repolarization and enhancing glucose oxidation. Recognition that a PDK-mediated metabolic shift contributes to contractile and ionic dysfunction in RVH offers insight into the pathophysiology and treatment of RVH.
Journal of Molecular Medicine 12/2009; 88(1):47-60. DOI:10.1007/s00109-009-0524-6 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroCT small animal 3D angiography has various applications such as tumor growth evaluation or quantification of the pulmonary arterial tree morphometry. Due to longer acquisition times using preclinical systems, classical contrast agents are of little use in-vivo. Novel contrast agents such as gold nanoparticles and iodinated liposomes offer a possible solution to this difficulty by preventing rapid renal excretion. To evaluate different contrast agents, we filled pieces of polymer tubing (20 AWG) with solutions of 10 mg Au/ml and 30 mg Au/ml home-made gold nanoparticles, commercial gold nanoparticle (120 mg Au/ml), commercial iodinated liposomes, 30% barium sulfate solution, 6% iodine contrast solution and 100% saline (PBS), respectively. Each piece of tubing was imaged in our microCT together with the PBS filled tubing using identical imaging settings. Upon quantification and comparison of the phantom data, we imaged mice after injection of contrast agent. We also acquired data from excised lungs filled with contrast agent and gelatin solution. The barium sulfate solution had the highest measured radiodensity, which was 8 times higher than PBS. The solution with only 6% of iodine contrast gave us an increased radiodensity of 1.5x. Among the in-vivo compounds, the liposomal contrast agent had the highest radiodensity with 5 times than PBS, where the gold nanoparticles (10 mg/ml) were almost indistinguishable from PBS, but the higher concentrations show improvements in contrast. Our comparison shows that the traditional contrast agents have a higher radiodensity than novel compounds currently under development. Angiography using barium sulfate is a terminal experiment where the iodine contrast agent is cleared too quickly by the kidneys to be useful in-vivo. The small contrast improvement of the gold nanoparticles indicate that further development is still necessary for the particles to by useful in 3D angiography. In contrast, liposomal contrast agents appear to -
have reached a state where they can successfully highlight vessels in-vivo.
[Show abstract][Hide abstract] ABSTRACT: As pulmonary artery obstruction results in proliferation of the bronchial circulation in a variety of species, we investigated this angiogenic response using single photon emission computed tomography (SPECT) and micro-CT.
After surgical ligation of the left pulmonary artery of rats, they were imaged at 10, 20, or 40 days post-ligation. Before imaging, technetium-labeled macroaggregated albumin ((99m)Tc MAA) was injected into the aortic arch (IA) labeling the systemic circulation. SPECT/micro-CT imaging was performed, the image volumes were registered, and activity in the left lung via the bronchial circulation was used as a marker of bronchial blood flow. To calibrate and to verify successful ligation, (99m)Tc MAA was subsequently injected into the left femoral vein (IV), resulting in accumulation within the pulmonary circulation. The rats were reimaged, and the ratio of the IA to the IV measurements reflected the fraction of cardiac output (CO) to the left lung via the bronchial circulation. Control and sham-operated rats were studied similarly.
The left lung bronchial circulation of the control group was 2.5% of CO. The sham-operated rats showed no significant difference from the control. However, 20 and 40 days post-ligation, the bronchial circulation blood flow had increased to 7.9 and 13.9%, respectively, of CO. Excised lungs examined after barium filling of the systemic vasculature confirmed neovascularization as evidenced by tortuous vessels arising from the mediastinum and bronchial circulation.
Thus, we conclude that SPECT/micro-CT imaging is a valuable methodology for monitoring angiogenesis in the lung and, potentially, for evaluating the effects of pro- or anti-angiogenic treatments using a similar approach.
European journal of nuclear medicine and molecular imaging 07/2008; 35(6):1124-32. DOI:10.1007/s00259-007-0684-y · 5.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bronchial circulation provides the lung with an oxygenated blood supply, derived from the aorta, whose primary purpose
under normal conditions is thought to be nourishment of the airway walls +Guyton, 1996-. However,the study of the bronchial
circulation and its development is particularly important with regard to lung tumors whose blood supply is via the bronchial
circulation rather than the pulmonary circulation +Hirsch, 2001, Ohta, 2002-. Thus, we have developed a rat model of bronchial
circulation angiogenesis,induced by complete occlusion of the left pulmonary artery. We have been observing the resulting
perfusion changes in the left lung using dual-modality SPECT/micro-CT imaging. The initial objective of this study is to develop
the necessary imaging system, protocol, and analysis methods for determining the time course of this angiogenesis, with the
subsequent goal of using this approach to study the particular angiogenic mechanisms that might be involved.
[Show abstract][Hide abstract] ABSTRACT: Small animal SPECT using low energy photons of I-125 and approaching resolutions of microscopic levels, imaging parameters such as pinhole edge penetration, detector blur, geometric response, detector and pinhole misalignment, and gamma photon attenuation and scatter can have increasingly noticeable and/or adverse effects on reconstructed image quality. Iterative reconstruction algorithms, the widelyaccepted standard for emission tomography, allow modeling of such parameters through a system matrix. For this Monte Carlo simulation study, non-uniform attenuation correction was added to the existing system model. The model was constructed using ray-tracing and further included corrections for edge penetration, detector blur, and geometric aperture response. For each ray passing through different aperture locations, this method attenuates a voxel's contribution to a detector element along the photon path, which is then weighted according to a pinhole penetration model. To lower the computational and memory expenses, symmetry along the detector axes and an incremental storage scheme for the system model were used. For evaluating the nonuniform attenuation correction method, 3 phantoms were designed of which projection images were simulated using Monte Carlo methods. The first phantom was used to examined skin artifacts, the second to simulate attenuation by bone, and the third to generate artifacts of an air-filled space surrounded by soft tissue. In reconstructions without attenuation correction, artifacts were observed with up to a 40% difference in activity. These could be corrected using the implemented method, although in one case overcorrection occurred. Overall, attenuation correction improved reconstruction accuracy of the radioisotope distribution in the presence of structural differences.
Proceedings of SPIE - The International Society for Optical Engineering 03/2007; 6510. DOI:10.1117/12.713568 · 0.20 Impact Factor