Paracrine regulation of endothelial cell motility and angiogenesis by deoxyribose-1-phosphate.

Angiogenesis (i.e. generation of new blood vessels from existing ones) is a central process in development, health, and tissue repair. During development, angiogenesis is responsible for the formation of the mature vascular system. In healthy individuals, angiogenesis ensures that the required levels of blood supply to tissues undergoing natural modification are maintained (e.g. ovaries, placenta and muscles). After injuries, such as burns, wounds, and bone fractures, or following the loss of tissue that results from diseases, or after surgical reconstructions, angiogenesis is the first critical step towards tissue repair. The regulation of angiogenesis is complex. It involves the interplay of different cell types and the participation of several signals and regulatory pathways. My and other's work suggested that deoxyribose-1-phosphate (dRP) stimulates angiogenesis by altering the characteristics of the cells lining the inner side of blood vessels (also known as endothelial cells). In particular, my most recent publication proved that dRP is released by platelets, small circulating cells mostly known for their role in stopping the bleeding that follows tissue injury by inducing blood clotting. In my experiments, the release of dRP by platelets induced changes in the ability of endothelial cells to move and it promoted the formation of new blood vessels in the chorioallantoic membrane, a tissue from chicken eggs that is commonly used to conveniently study blood vessel formation. These observations are completely novel and their importance lies in the fact that platelet-derived dRP may represent the link between the interruption of bleeding by blood clotting and the beginning of tissue repair. The biochemical nature of dRP is unusual for a pro-angiogenic signal (a small molecule derived from the metabolism of nucleic acids rather than a protein growth factor) and the understanding of its mechanism of action will have very important implications for our understanding of angiogenesis. The experiments proposed in this project will allow us to understand how dRP interacts with endothelial cells and how dRP stimulates the ability of endothelial cells to form new blood vessels. Based on the information obtained in our in vitro experimentation, the cellular processes underlying the stimulation of angiogenesis by dRP will also be studied in vivo with a chicken egg model and a mouse model. In summary, this project will significantly improve our understanding of angiogenesis and will open novel avenues for the development of therapeutic tools to improve tissue repair after injuries, degeneration or surgery.

The investigation will be performed in primary endothelial cells (from human umbilical vein and lung microvascular endothelium) and will make use of human and mouse platelets. Radiolabelled 5'-[3H]-dRP, subcellular fraction and radiolabelling detection will be used to investigate the fate of exogenous dRP in endothelial cells. Endothelial protein interacting with dRP will be identified using dRP-affinity chromatography followed by tandem mass spectrometry (MS/MS). Two-dimensional difference gel electrophoresis (2D-DIGE) coupled to MS/MS will be used to identify protein expression changes induced by dRP. Chemical inhibitors and small interfering RNA-mediated genetic ablation of specific endothelial ROS-generating enzymes will be used to identify the source of dRP-induced ROS in endothelial cells. The role of protein glycation will be assessed using specific antibodies, inhibitors of the Maillard reaction, and boronate affinity chromatography-MS/MS for the detection of glycated proteins. The role of heme oxygenase-1 (HO-1) and its signal transduction mechanism, including the potential involvement of biliverdin, carbon monoxide, hypoxia-induced factor 1 (HIF-1), nuclear factor-erythroid 2-related factor 2 (Nrf2), nuclear factor kappa B (NFkB), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), and interleukin-8 (IL-8) will be investigated with pharmacological inhibition, genetic silencing (siRNA), specific ELISA assays and inhibitory antibodies. A transgenic animal model characterised by reduced release of dRP by platelets (TP-/-UP-/-) will be used to assess the physiological role of dRP release by platelets in vivo using a mouse Matrigel implant vascularisation assay. The molecular mechanism underlying dRP-induced angiogenesis in vivo will be investigated with specific inhibitory tools in the same mouse Matrigel implant vascularisation assay and a chick chorioallantoic membrane (CAM) vascularisation assay.

Angiogenesis (i.e. formation of new blood vessel from existing ones) is essential for the generation and maintenance of the vascular system, which guarantees blood supply and ultimately survival of all tissues in the body. Angiogenesis is a driving process during development of the adult vascular system, and in adult life angiogenesis still occurs in mature tissues, such as ovaries (undergoing periodic growth and regression during the menstrual cycle), placenta (for the generation of the vascular network that allows the exchange of gas and nutrients between maternal and foetal blood), and muscles (where exercise-stimulated angiogenesis is responsible for the modulation of vascular density). Importantly, angiogenesis is fundamental for the formation of new vasculature from damaged blood vessels during the regeneration of tissues that follows injury, disease or surgery. The stimulation of angiogenesis is a key strategy for the treatment of incomplete healing following injury, disease or surgery, which is the cause of scarring, fracture non-unions, loss of functionality of body parts, pain, deformity, amputation and disability. Angiogenesis also plays a pivotal role in the progression of cancer, atherosclerosis and other inflammatory syndromes, which are all characterised by abnormal vascularisation. Because of the importance of angiogenesis for so many physiological processes, the study of the regulatory pathways that control angiogenesis is an important and timely endeavour that will have a relevant impact on healthcare. This project will provide key information on a novel stimulatory pathway of angiogenesis and its molecular mechanism. We showed that deoxyribose-1-phosphate (dRP) is released by platelets and is able to stimulate endothelial cell motility in vitro and angiogenesis in vivo. These observations will have implications for our understanding of cardiovascular homeostasis and tissue regeneration. This project will represent the first step in the development of regenerative treatments for defective or delayed tissue repair and might improve the quality of life of millions of people. Compared to pro-angiogenic treatments currently used for regenerative purposes (either cell preparations or growth factors), dRP has the advantage to be a small molecule that requires limited time and costs for preparation by chemical synthesis. Moreover, the chemical nature of dRP ensures better pharmacokinetic, dosability and selectivity than growth factors, which are known to activate a plethora of signalling pathways leading to a significant number of off-target effects. For example, dRP does not induce cell proliferation, a common off-target effect that limits the safety and efficacy of growth factors in medicine. In summary, this project will be a state-of-the-art biochemical and cell biological characterisation of deoxyribose-1-phosphate, a novel pro-angiogenic molecule with enormous applicability in regenerative medicine. This project has the potential to positively impact on society by providing a novel approach for the health care of conditions characterised by reduced tissue repair, such non-healing injuries, wound scarring, severe burns, ulcers and diabetic foot, bone non-unions or extensive surgical reconstructions.