Pro-Calcific Environment Impairs Ischaemia-Driven Angiogenesis.

Peripheral arterial disease (PAD) is characterised by accelerated arterial calcification and impairment in angiogenesis. Studies implicate vascular calcification as a contributor to PAD, but the mechanisms remain unclear. We aimed to determine the effect of calcification on ischaemia-driven angiogenesis. Human coronary artery endothelial cells (ECs) were treated with calcification medium (CM: CaCl2 2.7 mM, Na2PO4 2.0 mM) for 24 h and exposed to normoxia (5% CO2) or hypoxia (1.2% O2; 5% CO2 balanced with N2). In normoxia, CM significantly inhibited tubule formation and migration and upregulated calcification markers of ALP, BMP2, and Runx2. CM elevated levels of calcification-protective gene OPG, demonstrating a compensatory mechanism by ECs. CM failed to induce pro-angiogenic regulators VEGFA and HIF-1α in hypoxia and further suppressed the phosphorylation of endothelial nitric oxide synthase (eNOS) that is essential for vascular function. In vivo, osteoprotegerin-deficient mice (OPG-/-), a calcification model, were subjected to hind-limb ischaemia (HLI) surgery. OPG-/- mice displayed elevated serum alkaline phosphatase (ALP) activity compared to wild-type controls. OPG-/- mice experienced striking reductions in blood-flow reperfusion in both 8-week-old and 6-month-old mice post-HLI. This coincided with significant impairment in tissue ischaemia and reduced limb function as assessed by clinical scoring (Tarlov). This study demonstrated for the first time that a pro-calcific environment is detrimental to ischaemia-driven angiogenesis. The degree of calcification in patients with PAD can often be a limiting factor with the use of standard therapies. These highly novel findings require further studies for full elucidation of the mechanisms involved and have implications for the development of therapies to suppress calcification in PAD.

Ultrathin monolithic 3D printed optical coherence tomography endoscopy for preclinical and clinical use.

Preclinical and clinical diagnostics increasingly rely on techniques to visualize internal organs at high resolution via endoscopes. Miniaturized endoscopic probes are necessary for imaging small luminal or delicate organs without causing trauma to tissue. However, current fabrication methods limit the imaging performance of highly miniaturized probes, restricting their widespread application. To overcome this limitation, we developed a novel ultrathin probe fabrication technique that utilizes 3D microprinting to reliably create side-facing freeform micro-optics (<130 µm diameter) on single-mode fibers. Using this technique, we built a fully functional ultrathin aberration-corrected optical coherence tomography probe. This is the smallest freeform 3D imaging probe yet reported, with a diameter of 0.457 mm, including the catheter sheath. We demonstrated image quality and mechanical flexibility by imaging atherosclerotic human and mouse arteries. The ability to provide microstructural information with the smallest optical coherence tomography catheter opens a gateway for novel minimally invasive applications in disease.

Plaque Calcification: Do Lipoproteins Have a Role?

Vascular calcification (VC) is strongly associated with all-cause mortality and is an independent predictor of cardiovascular events. Resulting from its complex, multifaceted nature, targeted treatments for VC have not yet been developed. Lipoproteins are well characterized in the pathogenesis of atherosclerotic plaques, leading to the development of plaque regressing therapeutics. Although their roles in plaque progression are well documented, their roles in VC, and calcification of a plaque, are not well understood. In this review, early in vitro data and clinical correlations suggest an inhibitory role for HDL (high-density lipoproteins) in VC, a stimulatory role for LDL (low-density lipoprotein) and VLDL (very low-density lipoprotein) and a potentially causal role for Lp(a) (lipoprotein [a]). Additionally, after treatment with a statin or PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitor, plaque calcification is observed to increase. With the notion that differing morphologies of plaque calcification associate with either a more stable or unstable plaque phenotype, uncovering the mechanisms of lipoprotein-artery wall interactions could produce targeted therapeutic options for VC.

Effects of ovarian disaggregation on adult murine follicle yield and viability.

Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 folliclesmg-1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n=7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590UmL-1) or animal origin-free collagenase IV (AOF) at 590 or 1180UmL-1. Isolated follicles were stained with 4',6'-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17±10 folliclesmg-1 ovarian tissue). All disaggregation methods released more secondary follicles (86±20 per ovary; P<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.