Martin Butcher

Effect of Leptin on Vascular Calcification in Apolipoprotein E–Deficient Mice

Objective—The adipocytokine leptin has been proposed to increase cardiovascular risk in both obese and diabetic individuals. In the current study, therefore, we used apoE-deficient mice to examine the effects of leptin on both lesion size and calcification. Methods and Results—Mice were treated with once daily intraperitoneal injections of leptin (125 &mgr;g/mouse/d) for 2 months. The mice were then euthanized, and sections of the aortic root and thoracic aorta analyzed histomorphometrically. Measurements of lesion size and surface area occupied by atherosclerotic lesions did not reveal any differences between nontreated and leptin-treated animals. However, von Kossa staining of the aortic root demonstrated an 8.3±2.0-fold increase in lesion calcification as well as a 2.5±0.6-fold increase in valvular calcification in those animals treated with leptin. In addition, the percent total lesion area demonstrating ALP-positive staining was 5.4±2.1-fold greater in leptin-treated mice when compared to nontreated control mice. This increase in ALP staining was also accompanied by an increase in the expression of the osteoblast-specific markers, osteocalcin, and osteopontin. Conclusions—Based on these observations, we conclude that leptin may increase cardiovascular risk by promoting osteogenic differentiation and thus vascular calcification.

Oxidized low‐density lipoprotein promotes osteoblast differentiation in primary cultures of vascular smooth muscle cells by up‐regulating Osterix expression in an Msx2‐dependent manner

We have previously shown that oxidized low‐density lipoproteins (oxLDLs) act synergistically with β‐glycerophosphate to induce the osteogenic differentiation of primary bovine aortic smooth muscle cells (BASMCs). In the present study, we attempt to resolve the mechanism responsible for this effect by examining the expression of several osteoblast‐specific transcription factors. Thus, by culturing BASMCs in the absence or presence of β‐glycerophosphate and/or oxLDL, we demonstrate that β‐glycerophosphate induces both Runx2 and Osterix (Osx) expression. In contrast, oxLDL has no effect on Runx2 expression but rather it enhances β‐glycerophosphate‐induced osteoblast differentiation by further up‐regulating Osx expression. In an attempt to elucidate the mechanism responsible for this latter effect, we examined the ability of oxLDL to affect Msh homeobox 2 (Msx2) expression. Similar to its effect on Osx expression, oxLDL was found to synergistically enhance β‐glycerophosphate‐induced Msx2 expression in an extracellular signal‐regulated kinase 1 and 2 (Erk 1 and 2)‐dependent manner. Furthermore, oxLDLs ability to enhance both β‐glycerophosphate‐induced Osx expression and alkaline phosphatase activity was prevented when the BASMCs were first transfected with Msx2‐specific siRNA. Taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteoblast differentiation and vascular calcification in vivo. J. Cell. Biochem. 112: 581–588, 2011.

Oxidized low-density lipoprotein acts synergistically with β-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells

Previous studies have localized osteoblast specific markers to sites of calcified atherosclerotic lesions. We therefore decided to use an established in vitro model of vascular calcification in order to confirm earlier reports of oxidized low‐density lipoprotein (oxLDL) promoting the osteogenic differentiation of vascular smooth muscle cells. Treatment of primary bovine aortic smooth muscle cells (BASMCs) with β‐glycerophosphate was found to induce a time‐dependent increase in osteoblast differentiation. In contrast, no effect was seen when BASMCs were cultured in the presence of oxLDL alone. However, when the BASMCs were cultured in the presence of both β‐glycerophosphate and oxLDL, β‐glycerophosphates ability to induce osteoblast differentiation was significantly enhanced. In an attempt to resolve the mechanism by which this effect was occurring, we examined the effect of β‐glycerophosphate and oxLDL on several pathways known to be critical to the differentiation of osteoblasts. Surprisingly, β‐glycerophosphate alone was found to enhance Osterix (Osx) expression by inducing both Smad 1/5/8 activation and Runx2 expression. In contrast, oxLDL did not affect either Smad 1/5/8 activation or Runx2 activation but rather, it enhanced both β‐glycerophosphate‐induced Osx expression and osteoblast differentiation in an extracellular signal‐regulated kinase 1 and 2 (Erk 1 and 2) ‐dependent manner. When taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteogenic differentiation and vascular calcification in vivo. J. Cell. Biochem. 105: 185–193, 2008.

Structure and expression of the glycoprotein gene of Chandipura virus.

A cDNA copy of the mRNA for the glycoprotein G of Chandipura virus, a rhabdovirus, has been cloned, sequenced, and expressed in mammalian cells. The deduced amino acid sequence of G shows that the encoded protein is a typical transmembrane glycoprotein of 524 amino acids containing a cleavable amino-terminal signal peptide, two potential N-linked glycosylation sites, a hydrophobic membrane anchor domain near the carboxy terminus, and a cytoplasmic domain at the carboxy terminus. Somewhat unusual is the appearance of two charged amino acid residues, aspartate and arginine, within the putative membrane anchor sequence. Expression of the G gene in COS cells resulted in production of a glycosylated protein of mol wt 71,000 which was recognized by anti-Chandipura antibodies. Like the viral G protein, the expressed G contained covalently linked palmitic acid. However, unlike its vesicular stomatitis virus (Indiana serotype) counterpart, the Chandipura G protein has no potential palmitate-accepting cysteine residue within its cytoplasmic domain. Thus, the covalent attachment of fatty acid to this molecule may occur at one or both of the cysteines within the membrane anchor domain. The G protein was intracellularly transported to the cell surface and could induce cell fusion at low pH, showing that the expressed G protein was biologically active.

Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3β.

In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 μg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3β on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a >2-fold increase in GSK-3β activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3β activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

Differential effects of heparin and low molecular weight heparin on osteoblastogenesis and adipogenesis in vitro

We have previously demonstrated that heparin produces cancellous bone loss in rats due in part to a decrease in the number of osteoblasts lining the trabecular bone surface. In the present study, we use a stromal-derived cell culture system together with measurements of alkaline phosphatase (ALP) activity, to compare the effects of heparin and the low molecular weight heparin (LMWH), Fragmin, on osteoblast differentiation in vitro. In addition, we examined the possibility that both heparin and LMWH can induce adipogenesis in our stromal cell culture system. Both heparin and LMWH were found to produce a statistically significant (P < 0.01) and concentration-dependent decrease in the number of osteoblasts while increasing the number of adipocytes. When the effects of gravimetrically equivalent amounts of heparin and LMWH were compared, heparin had a 4-fold greater effect than LMWH. In contrast to heparin, N-desulfated heparin was found to have minimal effects on both osteoblast and adipocyte differentiation indicating that the heparin effect is not only chain-length dependent but also charge-dependent. The observation that LMWH has less of an effect on bone formation than heparin is compatible with the results of clinical trials indicating that LMWH produces less bone loss after long-term administration.

Heparin Synergistically Enhances Interleukin-11 Signaling through Up-regulation of the MAPK Pathway

Using an animal model of heparin-induced osteoporosis we previously demonstrated that heparin causes bone loss, in part, by increasing osteoclast number and activity. Furthermore, we found that, although heparin alone has no effect, it is able to synergistically enhance Interleukin-11 (IL-11)-induced signal transducer and activator of transcription 3 (STAT3) activation and thus increase osteoclast formation in vitro. In the present study, we examine the effect of various serine kinase inhibitors on the ability of heparin to act synergistically with IL-11. Inhibition of the c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or the phosphatidylinositol 3-kinase pathways had no effect on the ability of heparin to promote either IL-11-induced STAT3·DNA complex formation or osteoclast formation in vitro. In contrast, PD098059, a MAPK kinase inhibitor, completely abolished the synergy between heparin and IL-11. In an attempt to resolve the mechanism by which this was occurring, we examined the effect of heparin on STAT3 Ser-727 phosphorylation and extracellular signal-regulated kinases 1 and 2 (Erk1/2) activation, either in the presence or absence of IL-11. Heparin alone was found to have no effect on Ser-727 phosphorylation, nor did heparin alter the phosphorylation status of Ser-727 in the presence of IL-11. Heparin was, however, found to increase Erk1/2 activation in both a time- and dose-dependent manner. When taken together, these findings suggest that heparin enhances IL-11-induced STAT3 activation and thus osteoclast formation, by a mechanism that is independent of STAT3 Ser-727 phosphorylation but that involves up-regulation of the MAPK pathway.

Expression from cloned DNA of biologically active glycoprotein C of herpes simplex virus type 1 in mammalian cells.

A DNA fragment of the herpes simplex virus type 1 genome encoding glycoprotein C (gC-1) has been cloned into different eukaryotic expression vectors for transient and stable expression of the glycoprotein in a number of cell lines. All of these expression vectors use a non-HSV promoter, such as the adenovirus major late promoter or murine leukemia virus long terminal repeat promoter to express gC-1 in COS and CHO cells or 3T3 cells. The gC-1 protein synthesized was fully glycosylated with both N- and O-linked oligosaccharides. Synthesis of the mature 120K gC-1 glycoprotein involved partially glycosylated 100K and 105K proteins and the non-glycosylated 70K protein as intermediate molecules. Immunofluorescence studies showed that the expressed gC-1 was localized intracellularly in the nuclear envelope as well as on the cell surface. The expressed gC-1 was biologically active and could act as a receptor for the complement component C3b in the absence of other HSV proteins.

Ex vivo-expanded NK cells from blood and ascites of ovarian cancer patients are cytotoxic against autologous primary ovarian cancer cells

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.

Targeted Knockdown of Tissue Factor in B16F10 Melanoma cells suppresses their Ability to Metastasize to Bone and cause cancellous Bone Loss

In this study, we use a well-defined mouse model to examine tissue factor’s (TF) role in osteolytic bone metastasis. C57BL/6 mice received either mock siRNA-transfected or TF-specific siRNA-transfected B16F10 melanoma cells by left ventricular injection. A third group served as an age-matched control and did not receive any tumour cells. The effect on tumour burden and bone strength was then determined 14 days later by using bone histomorphometry and biomechanical testing. Based on histomorphometric analysis of the femurs, mice receiving TF-specific siRNA-transfected tumour cells had significantly reduced tumour burden as compared to those from mice that received mock siRNA-transfected tumour cells (2.20 ± 0.58% vs. 9.18 ± 2.20%). Furthermore, the femurs from mice receiving TF siRNA-transfected tumour cells displayed decreased osteoclast surface and consequently, increased cancellous bone volume and strength when compared to those isolated from mice that were injected with mock-transfected tumour cells. More importantly, no differences in osteoclast surface or cancellous bone volume and strength were observed when the femurs of mice that received TF siRNA-transfected tumour cells were compared to control mice that did not receive tumour cells. Based on these findings, we conclude that the expression of TF by tumour cells promotes their ability to metastasize to bone, thereby facilitating tumour cell—induced cancellous bone loss.