Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

Engineered Ovarian Cancer Cell Lines for Validation of CAR T Cell Function.

A set of genetically engineered isogenic cell lines is developed to express either folate receptor alpha or mesothelin, and a control cell line negative for both antigens. These cell lines also express fluorescent and bioluminescent reporter transgenes. The cell lines are used to authenticate specificity and function of a T-cell biofactory, a living vector that is developed to express proportionate amounts of engineered proteins upon engaging with disease cells through their specific antigenic biomarkers. The engineered cell lines are also used to assess the cytolytic function and specificity of primary T cells engineered with chimeric antigen receptors; and the specificity of monoclonal antibodies. The strategy described can be used to generate other cell lines to present different disease-specific biomarkers for use as quality control tools.

Regulation of lysyl oxidase in vascular cells: lysyl oxidase as a new player in cardiovascular diseases.

Lysyl oxidase (LOX) plays a crucial role in the maintenance of extracellular matrix stability and could participate in vascular remodelling associated with cardiovascular diseases. Evidence from in vitro and in vivo studies shows that LOX downregulation is associated with the endothelial dysfunction characteristic of earlier stages of the atherosclerotic process. Conversely, upregulation of this enzyme in vascular cells could induce neointimal thickening in atherosclerosis and restenosis. In fact, LOX is chemotactic for vascular smooth muscle cells and monocytes, is modulated by proliferative stimulus in these cells, and could control other cellular processes such as gene expression and cell transformation. Furthermore, it is conceivable that LOX downregulation could underlie plaque instability and contribute to the destructive remodelling that takes place during aneurysm development. Overall, LOX could play a key role in vascular homeostasis and, hence, it emerges as a new player in cardiovascular diseases. This review addresses the experimental evidence related to the role of LOX in vascular disorders and the potential benefits of controlling its expression and function.

A compact synthetic pathway rewires cancer signaling to therapeutic effector release.

An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.

Lysyl oxidase and endothelial dysfunction: mechanisms of lysyl oxidase down-regulation by pro-inflammatory cytokines.

Lysyl oxidase (LOX) plays a pivotal role in extracellular matrix (ECM) maturation. Furthermore, novel biological functions has been ascribed to LOX, among them cell differentiation, migration, transformation and regulation of gene expression. In this context, it has been suggested that abnormalities of LOX expression could underlie the development of multiple pathological processes including cardiovascular diseases. LOX seems to be crucial in the preservation of endothelial barrier function. In fact, accumulating evidences suggest a role of this enzyme in atherogenesis and endothelial dysfunction triggered by atherosclerotic risk factors and pro-inflammatory cytokines. Indeed, cytokines such as tumour necrosis factor-alpha (TNF-alpha) modulate vascular LOX expression. This cytokine decreases LOX expression and activity in endothelial cells through a transcriptional mechanism that involves TNF receptor-2 and protein kinase C activation. Interestingly, in vivo studies reveal that TNF-alpha causes a down-regulation of vascular LOX expression. Thus, LOX down-regulation seems to be associated to the endothelial dysfunction elicited by multiple pathological factors. LOX rises as a promising target gene for the development of therapeutic strategies in the treatment of cardiovascular diseases.

Hypoxia-induced ROS signaling is required for LOX up-regulation in endothelial cells.

The adaptive response of endothelial cells to hypoxia involves a substantial remodeling of extracellular matrix (ECM). In endothelial cells hypoxia up-regulates lysyl oxidase (LOX), a key enzyme in ECM assembly, relevant to vascular homeostasis. However, the mechanism underlying this response has not been established. Hypoxia up-regulated LOX expression in endothelial cells (HUVEC and BAEC) and concomitantly increased LOX enzymatic activity. This effect was independent of autocrine factors released by hypoxic cells and relies on a transcriptional mechanism. Both mTOR blockade and HIF-1alpha knockdown slightly prevented LOX up-regulation by hypoxia, suggesting that HIF-1alpha is only partially responsible for this effect. In fact, serial promoter deletion and mutagenesis studies indicated a limited contribution of the previously described hypoxia response element (-75 bp). Interestingly, Smad over-expression further increased LOX transcriptional activity in endothelial cells exposed to hypoxia. Moreover, the increase in LOX expression triggered by hypoxia was significantly reduced by reactive oxygen species (ROS) inhibitors. Thus, our data support a role of Smad signaling and ROS in the up-regulation of LOX by hypoxia in endothelial cells.

Statins normalize vascular lysyl oxidase down-regulation induced by proatherogenic risk factors.

AIMS: Statins are lipid-lowering drugs widely used in the management of vascular diseases. Clinical and experimental evidence suggest that statins improve endothelial function by both cholesterol-lowering-dependent and -independent mechanisms. We have previously shown that endothelial dysfunction induced by risk factors and proinflammatory cytokines is associated with down-regulation of lysyl oxidase (LOX), a key enzyme modulating extracellular matrix maturation and vascular integrity. Our aim was to analyse whether statins could normalize LOX expression impaired by proatherogenic risk factors. METHODS AND RESULTS: We observed that pharmacological concentrations of statins (atorvastatin and simvastatin) modulated LOX transcriptional activity, counteracting the down-regulation of LOX (at the mRNA, protein, and activity level) caused by tumour necrosis factor-alpha (TNFalpha) in porcine, bovine, and human aortic endothelial cells. Geranylgeraniol but not farnesol reversed this effect, suggesting the involvement of geranylgeranylated proteins. In accordance, inhibitors of RhoA/Rho kinase also counteracted LOX down-regulation caused by TNFalpha, and over-expression of a RhoA dominant-negative mutant mimicked statin effects. Statins were also able to counteract the decrease in LOX expression produced by atherogenic concentrations of LDL by a similar mechanism and to partially prevent the increase in endothelial permeability elicited by these lipoproteins. Finally, in the in vivo porcine model of hypercholesterolaemia, we observed that statins abrogated the reduction of vascular LOX expression triggered by high plasma levels of LDL. CONCLUSION: These data indicate that statins normalize vascular LOX expression altered by atherogenic risk factors through a RhoA/Rho kinase-dependent mechanism. Thus, modulation of LOX by statins could contribute to vascular protection and to the cardiovascular risk reduction achieved by this therapy.