Metformin directly suppresses atherosclerosis in normoglycaemic mice via haematopoietic adenosine monophosphate-activated protein kinase.

AIMS: Atherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque haemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and activating transcription factor 1 (ATF1). The antidiabetic drug metformin may also activate AMPK-dependent signalling. Hypothesis: Metformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, thereby suppresses atherogenesis. METHODS AND RESULTS: Normoglycaemic Ldlr-/- hyperlipidaemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (P < 5 × 10-11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK [analysis of variance (ANOVA), P < 0.03]. Metformin at a clinically relevant concentration (10 µM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1, and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin-induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin-induced lesional macrophage expression of p-AMPK, p-ATF1, and downstream M2-like protective effects. CONCLUSION: Metformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidaemic mice via haematopoietic AMPK.

FTI-277 inhibits smooth muscle cell calcification by up-regulating PI3K/Akt signaling and inhibiting apoptosis.

BACKGROUND: Vascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs. RESULTS: We demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) in vitro, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an ex vivo model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure. CONCLUSIONS: Together, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.

Near Infrared Fluorescence (NIRF) Molecular Imaging of Oxidized LDL with an Autoantibody in Experimental Atherosclerosis.

We aimed to develop a quantitative antibody-based near infrared fluorescence (NIRF) approach for the imaging of oxidized LDL in atherosclerosis. LO1, a well- characterized monoclonal autoantibody that reacts with malondialdehyde-conjugated LDL, was labeled with a NIRF dye to yield LO1-750. LO1-750 specifically identified necrotic core in ex vivo human coronary lesions. Injection of LO1-750 into high fat (HF) fed atherosclerotic Ldlr(-/-) mice led to specific focal localization within the aortic arch and its branches, as detected by fluorescence molecular tomography (FMT) combined with micro-computed tomography (CT). Ex vivo confocal microscopy confirmed LO1-750 subendothelial localization of LO1-750 at sites of atherosclerosis, in the vicinity of macrophages. When compared with a NIRF reporter of MMP activity (MMPSense-645-FAST), both probes produced statistically significant increases in NIRF signal in the Ldlr(-/-) model in relation to duration of HF diet. Upon withdrawing the HF diet, the reduction in oxLDL accumulation, as demonstrated with LO1-750, was less marked than the effect seen on MMP activity. In the rabbit, in vivo injected LO1-750 localization was successfully imaged ex vivo in aortic lesions with a customised intra-arterial NIRF detection catheter. A partially humanized chimeric LO1-Fab-Cys localized similarly to the parent antibody in murine atheroma showing promise for future translation.

PARP-14 combines with tristetraprolin in the selective posttranscriptional control of macrophage tissue factor expression.

Tissue factor (TF) (CD142) is a 47 kDa transmembrane cell surface glycoprotein that triggers the extrinsic coagulation cascade and links thrombosis with inflammation. Although macrophage TF expression is known to be regulated at the RNA level, very little is known about the mechanisms involved. Poly(adenosine 5'-diphosphate [ADP]-ribose)-polymerase (PARP)-14 belongs to a family of intracellular proteins that generate ADP-ribose posttranslational adducts. Functional screening of PARP-14-deficient macrophages mice revealed that PARP-14 deficiency leads to increased TF expression and functional activity in macrophages after challenge with bacterial lipopolysaccharide. This was related to an increase in TF messenger RNA (mRNA) stability. Ribonucleoprotein complex immunoprecipitation and biotinylated RNA pull-down assays demonstrated that PARP-14 forms a complex with the mRNA-destabilizing protein tristetraprolin (TTP) and a conserved adenylate-uridylate-rich element in the TF mRNA 3' untranslated region. TF mRNA regulation by PARP-14 was selective, as tumor necrosis factor (TNF)α mRNA, which is also regulated by TTP, was not altered in PARP-14 deficient macrophages. Consistent with the in vitro data, TF expression and TF activity, but not TNFα expression, were increased in Parp14(-/-) mice in vivo. Our study provides a novel mechanism for the posttranscriptional regulation of TF expression, indicating that this is selectively regulated by PARP-14.

Axl tyrosine kinase protects against tubulo-interstitial apoptosis and progression of renal failure in a murine model of chronic kidney disease and hyperphosphataemia.

Chronic kidney disease (CKD) is defined as the progressive loss of renal function often involving glomerular, tubulo-interstitial and vascular pathology. CKD is associated with vascular calcification; the extent of which predicts morbidity and mortality. However, the molecular regulation of these events and the progression of chronic kidney disease are not fully elucidated. To investigate the function of Axl receptor tyrosine kinase in CKD we performed a sub-total nephrectomy and fed high phosphate (1%) diet to Axl+/+ and Axl-/- mice. Plasma Gas6 (Axl' ligand), renal Axl expression and downstream Akt signalling were all significantly up-regulated in Axl+/+ mice following renal mass reduction and high phosphate diet, compared to age-matched controls. Axl-/- mice had significantly enhanced uraemia, reduced bodyweight and significantly reduced survival following sub-total nephrectomy and high phosphate diet compared to Axl+/+ mice; only 45% of Axl-/- mice survived to 14 weeks post-surgery compared to 87% of Axl+/+ mice. Histological analysis of kidney remnants revealed no effect of loss of Axl on glomerular hypertrophy, calcification or renal sclerosis but identified significantly increased tubulo-interstitial apoptosis in Axl-/- mice. Vascular calcification was not induced in Axl+/+ or Axl-/- mice in the time frame we were able to examine. In conclusion, we identify the up-regulation of Gas6/Axl signalling as a protective mechanism which reduces tubulo-interstitial apoptosis and slows progression to end-stage renal failure in the murine nephrectomy and high phosphate diet model of CKD.

Col2a1 lineage tracing reveals that the meniscus of the knee joint has a complex cellular origin.

The knee joint consists of multiple interacting tissues that are prone to injury- and disease-related degeneration. Although much is known about the structure and function of the knee's constituent tissues, relatively little is known about their cellular origin and the mechanisms governing their segregation. To investigate the origin and segregation of knee tissues in vivo we performed lineage tracing using a Col2a1-Cre/R26R mouse model system and compared the data obtained with actual Col2a1 expression. These studies demonstrated that at E13.5 the interzone at the presumptive joint site forms when cells within the Col2a1-expressing anlagen cease expression of Col2a1 and not through cellular invasion into the anlagen. Later in development these interzone cells form the cruciate ligament and inner medial meniscus of the knee. At E14.5, after interzone formation, cells that had never expressed Col2a1 appeared in the joint and formed the lateral meniscus. Furthermore, cells with a Col2a1-positive expression history combined with the negative cells to form the medial meniscus. The invading cells started to express Col2a1 1 week after birth, resulting in all cells within the meniscus synthesizing collagen II. These findings support a model of knee development in which cells present in the original anlagen combine with invading cells in the formation of this complex joint.

Lineage tracing using matrilin-1 gene expression reveals that articular chondrocytes exist as the joint interzone forms.

We have developed a mouse in which the Cre recombinase gene has been targeted to exon 1 of the matrilin-1 gene (Matn1) to investigate the origins of articular chondrocytes and the development of the knee joint. Analysis of joints from offspring of Matn1-Cre/R26R crosses demonstrated that articular chondrocytes are derived from cells that have never expressed matrilin-1 whereas the remainder of the chondrocytes in the cartilage anlagen expresses matrilin-1. A band of chondrocytes adjacent to the developing interzone in the E13.5 day knee joint became apparent because these chondrocytes did not turn on expression of matrilin-1 in contrast to the other chondrocytes of the anlagen. The chondrocytes of the presumptive articular surface therefore appear to arise directly from a subpopulation of early chondrocytes that do not activate matrilin-1 expression rather than by redifferentiation from the flattened cells of the interzone. In addition, lineage tracing using both Matn1-Cre/R26R and Col2a1-Cre/R26R lines indicated that non-cartilaginous structures in the knee such as cruciate ligament, synovium and some blood vessels are formed by cells derived from the early chondrocytes of the anlagen.