Arteriosclerosis is an important age-dependent disease encompassing atherosclerosis, in- stent restenosis, pulmonary hypertension, and autologous bypass grafting. The accumulation of neointimal vascular smooth muscle (VSMC)-like cells is a critical event in the pathology of vascular disease leading to intimal-medial thickening (IMT) and vessel remodelling, and is considered an essential marker of subclinical arteriosclerotic disease. Their origin remains controversial, with several cell fate-mapping studies in mice indicating that they are derived from medial VSMCs, resident Nestin/S100β + vascular stem cells, and/or endothelial cells (ECs) following endothelial-mesenchymal transition (EndoMT). It is widely accepted that exposure to pathologic reactive oxygen species (ROS) generating risk factors is central to this pathology. The effective pathophysiological response within the vessel wall following vascular injury is endothelial cell apoptosis rendering the vascular endothelium dysfunctional. In the past few years, compelling evidence now suggests a role for the generation of endothelial-derived extracellular vehicles (EVs) as crucial regulators in transferring biological information, either locally or remotely, to initiate the proliferation, migration, and accumulation of VSMC-like cells within subclinical arteriosclerotic lesions. Early detection of these lesions represents an important diagnostic objective. In this context, the main focus of this study was to develop novel strategies that interrogate and discriminate these discrete cell populations and detect the key signalling molecules within endothelial-derived EVs that dictate their fate. Specifically, single-cell photonic analysis using broadband light (autofluorescence), Raman and Fourier Transform Intra Red (FTIR) spectral datasets from normal VSMCs and lesional cells derived from human vessels ex vivo, in addition to human-induced pluripotent stem cell (HiPSC) progenitors and their myogenic progeny in vitro, were analysed using supervised machine learning as a novel diagnostic platform for early detection of vascular phenotypes within lesions. Moreover, the characteristics and effects of endothelial-derived EVs on resident vascular stem cell fate following hyperglycaemic-induced endothelial dysfunction were assessed using rat and HiPSC models in vitro as a potential surrogate marker for early lesion formation. The data clearly demonstrates that single cell photonic analysis can successfully discriminate and predict vascular phenotypes within lesions. Furthermore, endothelial derived EVs following hyperglycaemic-induced endothelial dysfunction promote resident vascular stem cell myogenic differentiation, growth and migration in vitro. These characteristics may represent important surrogate biomarkers for detection of early subclinical arteriosclerosis.