Gareth D. Hyde

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 knees 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.

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.

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.

Pericytes: Adaptable Vascular Progenitors

Pericytes are traditionally defined as elongated cells with multiple processes which are embedded within a microvascular basement membrane in close apposition to endothelial cells. However, it is now realized that these cells have a more widespread distribution than originally thought as cells resembling pericytes have also been identified in larger arteries and veins. Pericytes play important roles in many physiological processes, including regulating blood flow, maintaining the structural integrity of vessel walls, and regulating endothelial cell proliferation and differentiation. However, this chapter will focus on one specific area of pericyte biology which is currently receiving considerable attention: the fact that these cells are adaptable progenitor cells. The evidence that pericytes closely resemble mesenchymal stromal/stem cells, both in terms of the markers they express and their potential to differentiate along many different lineages, will be discussed, as will our current understanding of the factors which regulate pericyte differentiation. Finally, recent studies showing the therapeutic potential of these cells for tissue repair and regeneration will be outlined.

3 FTI-277 inhibits vascular calcification by activating downstream Pi3k/Akt signalling and preventing apoptosis of vascular smooth muscle cells

Vascular calcification, which involves the osteogenic differentiation of vascular smooth muscle cells (VSMC), is a major contributor to morbidity and mortality in patients with atherosclerosis, diabetes and end-stage kidney disease. We have shown that nitrogen-containing bisphosphonates attenuate vascular calcification by inhibiting farnesylpyrophosphate synthase, thereby depleting cells of farnesylpyrophosphate and geranylgeranylpyrophosphate which are essential for the prenylation and activation of small GTPases. This study aims to determine whether vascular calcification is regulated by protein prenylation. We demonstrate that a farnesyl transferase inhibitor, FTI-277, significantly inhibits β-glycerophosphate-induced calcification of VSMC in a dose-dependent manner (p<0.001). Pre-incubation of VSMCs with FTI-277 (10 μM) inhibits Ras activation and markedly enhances serum-induced Akt phosphorylation in VSMC. To determine whether FTI-277 inhibits mineralisation by promoting PI3K/Akt signalling, VSMCs were induced to mineralise in the presence of wortmannin (a PI3K inhibitor, 100 nM), FTI-277 (10 μM) or both wortmannin and FTI-277. Controls were incubated in the presence of vehicle. Wortmannin markedly promotes mineral deposition by VSMC, whereas FTI-277 inhibits mineralisation. Some mineralisation was detected in cells incubated in the presence of both reagents, demonstrating that the effects of FTI-277 can be negated, at least partially, by preventing downstream PI3K signalling. Using apoptosis assays and western blotting, we also demonstrate that FTI-277 inhibits high-phosphate-induced apoptosis of human VSMC and activation of caspase 3. These studies demonstrate that FTI-277 inhibits VSMC mineralisation, at least in part, by activating downstream PI3K/Akt signalling and preventing apoptosis. Studies are in progress to determine whether FTI-277 also regulates the osteogenic differentiation of VSMC.

NEAR-INFRARED FLUORESCENCE (NIRF) WHOLE BODY AND INTRA-ARTERIAL MOLECULAR IMAGING OF OXIDIZED LDL IN EXPERIMENTAL ATHEROSCLEROSIS

Specific imaging of oxidized LDL in atherosclerosis could provide new approaches to detect vulnerable plaques at risk of rupture. To image oxidized LDL we generated LO1, an IgG3 monoclonal antibody against MDA-LDL, and labelled it without loss of function with a NIR fluorochrome (excitation 750nm

5 Gas6/Axl signalling supresses both osteogenic differentiation and apoptosis of vascular smooth muscle cells during phosphate-induced mineralisation

Vascular calcification is a major cause of morbidity and mortality; particularly in patients with end-stage kidney disease in whom elevated phosphate levels promote mineralisation. We previously identified that Gas6/Axl signalling inhibits vascular smooth muscle cell (VSMC) mineralisation. To understand the mechanism by which this inhibition occurs, we have now investigated its effect on key events during calcification, namely VSMC osteogenic differentiation and apoptosis. In short-term assays addition of increasing concentrations of phosphate (0.9 €“ 3.4 mM) results in increased VSMC death. Treatment with Gas6 (400 ng/ml) reduces VSMC death and the amount of active caspase-3, demonstrating that Gas6 prevents phosphate-induced VSMC apoptosis. VSMC mineralisation was induced by adding 5 mM β-glycerophosphate to the culture medium. To determine the temporal pattern of expression of osteogenic markers during mineralisation, RNA was isolated every 2–3 days and qPCR was performed. These studies demonstrated that osteogenic transcription factors (Msx2, Twist and Runx2) are transiently upregulated during the early stages of calcium deposition by VSMC. To investigate the effect of Gas6/Axl signalling on VSMC osteogenic differentiation, Axl was overexpressed in VSMC using lentiviruses and Gas6 (100 ng/ml) was added. This resulted in a reduction in osteogenic marker expression and levels of active caspase-3, increased α smooth muscle actin expression and reduced calcium deposition. This study demonstrates that Axl/Gas6 signalling suppresses both VSMC osteogenic differentiation and apoptosis during phosphate-induced mineralisation and further highlights the pathway as a potential therapeutic target for the treatment of vascular calcification.