In general, hitherto, all scholarly illustrations of oxidative phosphorylation are schematically presented in two dimensions as in Fig.1, where the electron current is shown resulting in the transmembrane proton gradient driving the formation of ATP via the F1F0- ATPase [1]. In this hypothesis, the electrical gradient that results from electron transport induces a conformational change of the F1F0-ATPase coupled with a change of hydrogen bonding of a strategic shell of H2O molecules, favouring the thermodynamics of the reaction ADP with inorganic phosphate (Pi) to produce ATP. This hypothesis presents the whole coupling process of the mitochondrial inner membrane in three dimensions, essential for this particulate biochemistry [1,2].
Bone represents a well vascularized structure which is remodelled and renewed continuously. It consists of osteoblasts, osteoclasts and osteocytes which have a precise role in the context of endochondral and intramembranous ossification. While the link between the bone tissue, bone marrow and the haematopoiesis is crucial for the generation of progenitor bone forming cells, recent research indicates that bone physiology as well as bone healing, repair and regeneration is directly dependent on bone angiogenesis. Experimental research suggest that angiogenesis and osteogenesis are directly coupled through a mechanism in which type H endothelial cells have a role of paramount importance. Apart from type H endothelial cells, other molecular elements such as HIF and Notch as well as CD31-endomucin capillaries and miR-497∼195 endothelial clusters may have a significant role in angiogenesis-osteogenesis coupling. The number of type H endothelial cells decreases significantly in elderly and is paralleled by a significant drop in the supply of progenitor cells. This would explain the bone loss and the decreased bone regeneration potential seen in elderly patients. Overall, it is suggested that endothelial cells and bone angiogenesis would represent therapeutic targets in various pathological conditions characterized by bone loss and impaired regeneration.
The family of Notch proteins plays a key role in cell fate determination. Additionally, Notch proteins regulate critical functions of the endothelium, as well as other recruited supporting cells, in concert with other pathways. Despite significant advances in the field and extensive studies focused on elucidating this pathway, many questions remain regarding Notch activation and its upstream/downstream effects, with vascular biology constituting one area of particular interest. Here, we provide a brief description of the components and functions of the Notch pathway in vasculature, followed by a detailed compilation of recommended methods of evaluation in vitro and in vivo. We provide a rationale for key elements when choosing different approaches and controls, strengths and limitations, and essential considerations when providing a meaningful interpretation of results. Our aim is to describe a careful approach to assessing Notch function in endothelial cells, based on underlying principles, with the overall goal of obtaining physiologically relevant information that will enhance our understanding of this pathway and its role in vascular biology.
Background: Cryopreserved bone allografts are often used to reconstruct segmental bone defects. They are non-viable, which can result in infection, non-union and stress-fractures. We aimed to revascularize allografts in porcine and rat models using vascular endothelial growth factor (VEGF), combined with platelet derived growth factor (PDGF) administered through an adeno-associated viral vector. We report the development of vascular tumors resulting from this treatment. Methods: In two separate studies, an identical AAV.VEGF.PDGF vector was used to promote angiogenesis in cryopreserved bone allografts. In 8 Yucatan minipigs, a 3.5 cm segmental tibial defect was reconstructed with a matched allograft, revascularized by placement of a transfected arteriovenous (AV) bundle within the medullary canal. In another experiment, cryopreserved femoral bone allografts coated with AAV.VEGF.PDGF were placed across a 10 mm segmental femoral gap in 10 Lewis rats. Results: Vascular tumors developed in skin and subcutaneous tissues in 5 out of 8 pigs and all of the rats. Histology revealed changes essentially identical to those seen in pyogenic granuloma (lobular capillary hemangioma) in humans. Polymerase chain reaction (PCR) identified the sequence of human VEGF-DNA in all of the sampled tumor tissues. Conclusion: Recombinant AAV gene therapy used to promote angiogenesis in avascular bone risks the development of vascular cutaneous lesions. Gene therapy using an identical AAV.VEGF.PDGF vector should not be considered clinically until safe use can be demonstrated and.concerns regarding chromosomal integration, dose effect and species differences are addressed.
