Standardisation of microvascular imaging for unbiased assessment of reparative angiogenesis

Microvascular disease (MVD) urgently calls for new imaging and therapeutic approaches. We will test a modRNA therapy to help recovery from ischaemia and assess therapeutic results by unbiased analysis of microCT/PET imaging data. This multidisciplinary project will offer new means for effective diagnosis and therapy of MVD.

Microvascular Insufficiency represents a major cause of end-organ failure among elderly subjects and diabetic patients (Madeddu et al Circulation 2002;106(8):993-9; Front Biosci. 2007;12:2003-12). Gene-based angiogenesis therapy has suffered from poor control of dosage and duration, low gene transfer efficiency, the risk of genomic integration, and anti-viral immune responses. ModRNA, in which one or more nucleotides are replaced by modified nucleotides, may provide an effective means to control the spatial and temporal delivery of gene products for ischaemic tissue repair. Moreover, the lack of predictive imaging markers constitutes an important limitation in the management of patients with microvascular disease. Hence, the introduction of new methods for quantitative assessment of the imaging data is warranted. We propose a study with a three-fold aim: (1) to produce a modRNA version of the longevity-associated variant-BPIFB4 (LAV-BPIFB4), which as an adeno-associated viral vector-mediated gene transfer promoted reparative vascularization and improved laser Doppler blood flow recovery in a mouse model of limb ischaemia (Madeddu et al, Circ Res; 2015:117:333-45); (2) to assess the in vivo effects of LAV-BPIFB4 modRNA therapy on limb perfusion and angiogenesis by microCT/PET and immunohistochemistry of the muscle microvasculature; and (3) to use fractal geometry as an advanced method to decipher the complexity of the newly formed microvascular networks, with the long-term objective of developing a pioneering software for automated detection and functional interpretation of reparative angiogenesis. The aim of the numerical work is to test the hypothesis that the most efficient vascular network will result in the most rapid recovery.
WP1 (month 1-12). The student will acquire skills in vascular biology and animal modelling. He/she will produce the modRNA, according to published methods (Zangi et al, Nat Biotechnol; 31: 898–907). The transduction efficiency will be validated in vitro and in vivo to titrate the optimal dosage.
WP2 (month 13-36). The student will deliver the LAV-BPIFB4 modRNA or vehicle in mice with operative limb ischaemia, a model mastered in Madeddu’s laboratory. He/she will follow-up mice for 6 weeks with sequential laser Doppler and microCT/PET imaging supervised by Dr Paisey (PETIC, Cardiff). Animals will be sacrificed in groups at different stages post-ischaemia for assessment of capillary and arteriole density by IHC.
WP3 (month 24-36). Supervised by Dr Fraser and Cookson (Bath), the student will perform an unbiased fractal dimension analysis of the imaging data, an approach we have already employed for deciphering the complexity of angiogenesis in IHC preparations (Madeddu et al, Circ Res. 2010;107:283–93). This will be combined with 1D blood flow and 3D contrast agent diffusion modelling, to test the hypothesis that recovery is linked to the orderly remodelling of the microvascular network.