Christian Wietholt

Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

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 mm3 (P<0.05) with Mfn‐2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm3 (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.—Rehman, J., Zhang, H. J., Toth, P. T., Zhang, Y., Marsboom, G., Hong, Z., Salgia, R., Husain, A. N., Wietholt, C., Archer, S. L. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. FASEB J. 26, 2175‐2186 (2012). www.fasebj.org

The inhibition of pyruvate dehydrogenase kinase improves impaired cardiac function and electrical remodeling in two models of right ventricular hypertrophy: resuscitating the hibernating right ventricle

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 O2-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 QTc 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.

Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation

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.

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

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.

Lung 18F-Fluorodeoxyglucose Positron Emission Tomography for Diagnosis and Monitoring of Pulmonary Arterial Hypertension

RATIONALE 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). OBJECTIVES 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. METHODS 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. MEASUREMENTS AND MAIN RESULTS 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. CONCLUSIONS 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.

A Central Role for CD68(+) Macrophages in Hepatopulmonary Syndrome: Reversal by Macrophage Depletion

RATIONALE 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. OBJECTIVES We hypothesized that macrophages are central to HPS and investigated the therapeutic potential of macrophage depletion. METHODS 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). MEASUREMENTS AND MAIN RESULTS 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. CONCLUSIONS 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.

A novel functional CT contrast agent for molecular imaging of cancer

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.

AuNP-DG: Deoxyglucose-Labeled Gold Nanoparticles as X-ray Computed Tomography Contrast Agents for Cancer Imaging

PurposeTo 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.ProceduresGold 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.ResultsSignificant 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.ConclusionsResults 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.

Role of RANK-RANKL-OPG axis in cranial suture homeostasis.

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 &bgr;, minimal attention has been directed toward osteoclast function in cranial suture biology. Receptor activator of nuclear factor &kgr;B (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 vanadyl chelate bis(acetylacetonato)oxovanadium(IV) increases the fractional uptake of 2-(fluorine-18)-2-deoxy-D-glucose by cultured human breast carcinoma cells

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.