Metabolic Regulation of mTOR Activation and Endoplasmic Reticulum Stress in the Heart

When subjected to increased workload, the heart responds metabolically by increasing its reliance on glucose and structurally by increasing the size of myocytes. Whether changes in metabolism regulate the structural remodeling process is unknown. A likely candidate for a link between metabolism and growth in the heart is the mammalian target of rapamycin (mTOR), which couples energy and nutrient metabolism to cell growth. Recently, sustained mTOR activation has also been implicated in the development of endoplasmic reticulum (ER) stress. We explored possible mechanisms by which acute metabolic changes in the hemodynamically stressed heart regulate mTOR activation, ER stress and cardiac function in the ex vivo isolated working rat heart. Doubling the heart’s workload acutely increased rates of glucose uptake beyond rates of glucose oxidation. The concomitant increase in glucose 6-phosphate (G6P) was associated with mTOR activation, endoplasmic reticulum (ER) stress and impaired contractile function. Both rapamycin and metformin restored glycolytic homeostasis, relieved ER stress and rescued contractile function. G6P and ER stress were also downregulated with mechanical unloading of failing human hearts. Taken together, the data support the hypothesis that metabolic remodeling precedes, triggers, and sustains structural remodeling of the heart and implicate a critical role for G6P in load-induced contractile dysfunction, mTOR activation and ER stress. In general terms, the intermediary metabolism of energy providing substrates provides signals for the onset and progression of hypertrophy and heart failure.

Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI2), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI2 biosynthesis substantially in humans. Because deletion of the PGI2 receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF1α by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI2 biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 ± 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.

Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.

Administration of interleukin 12 in combination with type II collagen induces severe arthritis in DBA/1 mice.

The induction of arthritis in DBA/1 mice usually requires immunization with the antigen type II collagen emulsified with Mycobacterium tuberculosis in oil. Here we describe that interleukin 12 (IL-12) can replace mycobacteria and cause severe arthritis of DBA/1 mice when administered in combination with type II collagen. Immunization of DBA/1 mice with type II collagen emulsified in oil alone resulted in a weak immune response, and only a few animals (10-30%) developed arthritis. Administration of IL-12 for 5 days simultaneously with each immunization strongly enhanced the anti-type II collagen immune response. Collagen-specific interferon gamma (IFN-gamma) synthesis by ex vivo activated spleen cells was enhanced 3- to 10-fold. IFN-gamma was almost completely produced by CD4+ T cells. Furthermore, the production of collagen-specific IgG2a and IgG2b antibodies was upregulated 10- to 100-fold. As a consequence, the incidence of arthritis in the group of mice immunized with collagen plus IL-12 was very high (80-100%). The developing arthritis was severe, involving approximately 50% of all limbs with strongly increased footpad thickness in most cases. Furthermore, histological examination revealed massive, mainly polymorphonuclear cell infiltration, synovial hyperplasia, cartilage and bone destruction, as well as new bone formation. In many cases, this resulted in the complete loss of joint structure. Neutralization of IFN-gamma in vivo prevented the development of arthritis in collagen-immunized and IL-12-treated mice. In conclusion, our data show that in vivo administered IL-12 can profoundly upregulate a T helper I-type autoimmune response, resulting in severe joint disease in DBA/1 mice.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Deletion of the Basement Membrane Heparan Sulfate Proteoglycan Type XVIII Collagen Causes Hypertriglyceridemia in Mice and Humans

Background: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. Methods and Findings: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. Conclusions: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

Ischaemic preconditioning improves proteasomal activity and increases the degradation of delta PKC during reperfusion

The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilon PKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection. Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished delta PKC translocation by 3.8-fold and increased epsilon PKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of delta PKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilon PKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilon PKC during IPC restored delta PKC levels at the mitochondria while decreasing epsilon PKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a delta PKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol. Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, delta PKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilon PKC.

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets

The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (Mempty set) are commonly drawn for functional studies. Here we define two Mempty set subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal Mempty set (LPM), contains approximately 90% of the PerC Mempty set in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical Mempty set surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal Mempty set (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of Mempty set heterogeneity and shed new light on PerC Mempty set diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC Mempty set.

Population balance model of in vivo neutrophil formation following bone marrow rescue therapy

In this paper, we develop a simple four parameter population balance model of in vivo neutrophil formation following bone marrow rescue therapy. The model is used to predict the number and type of neutrophil progenitors required to abrogate the period of severe neutropenia that normally follows a bone marrow transplant. The estimated total number of 5 billion neutrophil progenitors is consistent with the value extrapolated from a human trial. The model provides a basis for designing ex vivo expansion protocols.

Induction of CRP3/MLP expression during vein arterialization is dependent on stretch rather than shear stress

Aims Cysteine- and glycine-rich protein 3/muscle LIM-domain protein (CRP3/MLP) mediates protein-protein interaction with actin filaments in the heart and is involved in muscle differentiation and vascular remodelling. Here, we assessed the induction of CRP3/MLP expression during arterialization in human and rat veins. Methods and results Vascular CRP3/MLP expression was mainly observed in arterial samples from both human and rat. Using quantitative real time RT-PCR, we demonstrated that the CRP3/MLP expression was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1-14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer and later (28-90 days) it appeared more scattered in the vessel layers. Conclusion Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic conditions.