https://ir.ymlib.yonsei.ac.kr/bitstream/22282913/174897/1/T202000115.pdf

Non-invasive in vivo imaging of caspase-1 activation enables rapid and spatiotemporal detection of acute and chronic inflammatory disorders.

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications.

The skin is a recognized model to study normal and abnormal microcirculation, and the progressive development of non-invasive technologies, such as the laser Doppler flowmetry (LDF) and transcutaneous (tc) gasimetry reinforced that view. However, these technologies record oscillatory phenomena, complex in nature and difficult to interpret, which limits the in vivo utility of this information. The present study was designed to deepen our knowledge on these matters through the question how do LDF oscillatory components contribute to the regulation of microcirculatory function?. To answer this question the following objectives were established: (I) to develop methodologies that will allow to obtain functional microcirculatory parameters in vivo in healthy patients of different ages, and to characterize eventual changes related to ageing; (II) to find the best calculation tools to refine the LDF signal analysis; (III) to develop an animal model that allows us to test the best instruments developed, looking deeper, if possible, to the related phenomena; For the first objective two groups of healthy volunteers were studied ? one group of 35 healthy volunteers (22.1 ± 3.7 y.o.), and one other group of 30 volunteers, also healthy but older (50.8 ± 7.6 y.o.). In both groups the LDF signal was recorded following the application of three experimental protocols - passive leg raising (PLR), suprasystolic occlusion at the ankle level (SAO), and 100% oxygen breathing (OB). For the second objective three analytical tools were applied - the wavelet transform (WT), the detrended fluctuation analysis (DFA), and the multiscale entropy analysis (MSE). For the third objective, one group of 16 C57BL/6 mice was used (mean age 17 weeks old), submitted to a 100% oxygen breathing while anesthetized, and another group of 9 mice, 16 weeks old, submitted to an unilateral hindlimb ischemia, breathing 100% oxygen before and after procedure.

Contribución a la comprensión de la microcirculación periférica y a su regulación a través de los componentes oscilatórios de flujo medido por láser Doppler

Critical limb ischemia (CLI) is a common cause of high vascular morbidity and mortality. Monitoring the development and treatment response of hindlimb ischemia (HI) in an animal model enables a better understanding of the pathological mechanisms underlying CLI, and evaluation of the efficacy of novel therapeutic approaches. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue including extracellular matrix degradation and angiogenesis. Herein, a mouse HI model is established and subjected to noninvasive optical imaging with a novel and ultra-sensitive MMP activatable probe, termed MMP-P12, for analyzing the development and treatment response of HI. Our results show that angiogenesis development during HI was well correlated with MMP-2 activity alteration as examined by western blot, histological staining and MMP-P12 fluorescence signal recovery. Moreover, vascular endothelial growth factor (VEGF) mediated HI treatment was also monitored by MMP-P12. Up-regulated MMP-2 expression and an enhancement of angiogenesis were observed after VEGF treatment, which peaked at 7 days after the treatment. Overall, our results showed that MMP-2 plays an important role in the monitoring of angiogenesis during HI development and therapy. Application of MMP-P12 to visualize MMP-2 activity alteration can serve as a promising noninvasive optical imaging strategy to monitor angiogenesis and its response to therapy in CLI.

Noninvasive monitoring of the development and treatment response of ischemic hindlimb by targeting matrix metalloproteinase-2 (MMP-2)

Vascular endothelial growth factor receptor (VEGFR) and matrix metalloproteinase (MMP) are up-regulated in ischemic tissue and play pivotal roles in promoting angiogenesis. The purpose of the present study was to evaluate two fluorophore-conjugated peptide probes specific to VEGFR and MMP for dual-targeted in vivo monitoring of angiogenesis in a murine model of hindlimb ischemia. To this end, VEGFR-Probe and MMP-Probe were developed by conjugating distinct near-infrared fluorophores to VEGFR-binding and MMP substrate peptides, respectively. VEGFR-Probe exhibited specific binding to VEGFR on HUVECs, and self-quenched MMP-Probe produced strong fluorescence intensity in the presence of MMPs in vitro. Subsequently, VEGFR-Probe and MMP-Probe were successfully utilized for time course in vivo visualization of VEGFR or MMP, respectively. Simultaneous visualization provided information regarding the spatial distribution of these proteins, including areas of co-localization. This dual-targeted in vivo imaging approach will be useful for understanding the detailed mechanism of angiogenesis and for evaluating therapeutic angiogenesis.

In vivo monitoring of angiogenesis in a mouse hindlimb ischemia model using fluorescent peptide-based probes

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In vivo monitoring of angiogenesis in a mouse hindlimb ischemia model using fluorescent peptide-based probes