Cathepsin B is a lysosomal cysteine protease that plays an important role in cancer, atherosclerosis, and other inflammatory diseases. The suppression of cathepsin B can inhibit tumor growth. The overexpression of cathepsin B can be used for the imaging and photodynamic therapy (PDT) of cancer. PDT targeting of cathepsin B may have a significant potential for selective destruction of cells with high cathepsin B activity. We synthesized a cathepsin B-cleavable polymeric photosensitizer prodrug (CTSB-PPP) that releases pheophorbide a (Pha), an efficient photosensitizer upon activation with cathepsin B. We determined the concentration dependant uptake in vitro, the safety, and subsequent PDT-induced toxicity of CTSB-PPP, and ROS production. CTSB-PPP was cleaved in bone marrow cells (BMCs), which express a high cathepsin B level. We showed that the intracellular fluorescence of Pha increased with increasing doses (3–48 µM) and exerted significant dark toxicity above 12 µM, as assessed by MTT assay. However, 6 µM showed no toxicity on cell viability and ex vivo vascular function. Time-dependent studies revealed that cellular accumulation of CTSB-PPP (6 µM) peaked at 60 min of treatment. PDT (light dose: 0–100 J/cm2, fluence rate: 100 mW/cm2) was applied after CTSB-PPP treatment (6 µM for 60 min) using a special frontal light diffuser coupled to a diode laser (671 nm). PDT resulted in a light dose-dependent reduction in the viability of BMCs and was associated with an increased intracellular ROS generation. Fluorescence and ROS generation was significantly reduced when the BMCs were pre-treated with E64-d, a cysteine protease inhibitor. In conclusion, we provide evidence that CTSB-PPP showed no dark toxicity at low concentrations. This probe could be utilized as a potential imaging agent to identify cells or tissues with cathepsin B activity. CTSB-PPP-based PDT results in effective cytotoxicity and thus, holds great promise as a therapeutic agent for achieving the selective destruction of cells with high cathepsin B activity.
Interactions between macrophages, cardiac cells and the extracellular matrix are crucial for cardiac repair following myocardial infarction (MI). We hypothesized that cell-based treatments might modulate these interactions. After validating that bone marrow cells (BMC) associated with fibrin lowered the infarct extent and improved cardiac function, we interrogated the influence of fibrin, as a biologically active scaffold, on the secretome of BMC and the impact of their association on macrophage fate and cardiomyoblast proliferation. In vitro, BMC were primed with fibrin (F-BMC). RT-PCR and proteomic analyses showed that fibrin profoundly influenced the gene expression and the secretome of BMCs. Consequently, the secretome of F-BMC increased the spreading of cardiomyoblasts and showed an alleviated immunomodulatory capacity. Indeed, the proliferation of anti-inflammatory macrophages was augmented, and the phenotype of pro-inflammatory switched as shown by downregulated Nos2, Il6 and IL1b and upregulated Arg1, CD163, Tgfb and IL10. Interestingly, the secretome of F-BMC educated-macrophages stimulated the incorporation of EdU in cardiomyoblasts. In conclusion, our study provides evidence that BMC/fibrin-based treatment improved cardiac structure and function following MI. In vitro proofs-of-concept reveal that the F-BMC secretome increases cardiac cell size and promotes an anti-inflammatory response. Thenceforward, the F-BMC educated macrophages sequentially stimulated cardiac cell proliferation.
Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The study was supported by the Swiss National Science Foundation (SNF 310030_149986) attributed to MNG, the University of Fribourg and the Fonds Scientifique Cardiovasculaire FSC, Fribourg Hospital attributed to SC The spatiotemporal interaction of macrophages with the cardiac extracellular matrix, cardiomyocytes and non-cardiomyocytes has shown increasing interest in cardiac repair and regeneration. Due to their immunomodulatory capacities, cell-based therapies for myocardial infarction (MI) may influence macrophages fate. In addition, the use of biomaterials combined with cells is nowadays a recommended approach for cell-based therapies to fosters cell retention and survival. Depending on their composition and structure, scaffolds may modulate macrophage phenotypes. We interrogated the influence of fibrin, a biologically active scaffold, on the fate of cells including bone marrow cells (BMC), macrophages and cardiomyoblasts. Methods In vivo, two weeks post-MI induction, animals with an ejection fraction between 35-60% were either sham-operated animals or treated with an epicardial implantation of a BMC and fibrin. In vivo, two weeks post-MI induction, animals with an ejection fraction between 35-60% were either sham-operated animals or treated with an epicardial implantation of a BMC and fibrin. In vitro, non-polarized macrophages were differentiated toward either pro-inflammatory or anti-inflammatory phenotypes and stimulated with the conditioned medium of fibrin-primed BMC (F-BMC). Proteomic, cytokine levels quantification, and qPCR were performed. EdU incorporation and real time cell analysis assessed the effect of F-BMC on macrophages and cardiomyoblasts H9C2 proliferation. Results In vivo, epicardial implantation of Fibrin and BMC reduced the loss of cardiac function induced by MI and prevented the fibrotic scar expansion. After 4 and 12 weeks, the infarct content of CD68+ and CD206+ macrophages were similar in control and treated animals. To investigate acute effect, we performed in vitro assays. We showed that fibrin profoundly influenced gene expression and the secreted proteome of BMC, simultaneously upregulating both pro- and anti-inflammatory mediators. Furthermore, the conditioned medium from F-BMC significantly increased the proliferation of anti-inflammatory macrophages and modulated their gene expression and cytokines secretion. For example, F-BMC downregulated the expression of pro-inflammatory genes, in particular Nos2, Il6 and Ccl2/Mcp1. Anti-inflammatory genes such as Arg1, Tgfb and IL10 were significantly upregulated. Interestingly, anti-inflammatory macrophages educated by F-BMC stimulated EdU incorporation in H9C2 cardiomyoblasts. In conclusion, our study provides evidence that F-BMC secretome promoted the growth of anti-inflammatory macrophages, stimulated macrophage plasticity and altered the balance between the pro and anti-inflammatory macrophage subsets. F-BMC secretome favoured the mitogenic properties of anti-inflammatory macrophages promoting cardiac cell growth. In vivo, F-BMC treatment lowered the infarct extent and increased wall thickness and improved cardiac function.
Interactions between macrophages, cardiac cells and the extracellular matrix are crucial for cardiac repair following myocardial infarction (MI). The paracrine effects of cell-based treatments of MI might modulate these interactions and impact cardiac repair. The immunomodulatory capacity of the therapeutic cells is therefore of interest and could be modulated by the use of biomaterials. We first showed that bone marrow cells (BMC) associated with fibrin could treat MI. Then, we interrogated the influence of fibrin, as a biologically active scaffold, on the secretome of BMC and the impact of their association on macrophage fate and cardiomyoblast proliferation. Methods: In vivo, two weeks post-MI, rats were treated with epicardial implantation of BMC and fibrin or sham-operated. High-resolution echocardiography was performed to evaluate the heart function and structure changes after 4 weeeks. Histology and immunostaining were performed on harvested hearts. In vitro, BMC were first primed with fibrin. Second, non-polarized macrophages were differentiated toward either pro-inflammatory or anti-inflammatory phenotypes and stimulated with the conditioned medium of fibrin-primed BMC (F-BMC). Proteomic, cytokine levels quantification, and RT-PCR were performed. EdU incorporation and real-time cell analysis assessed cell proliferation. Results: The epicardial implantation of fibrin and BMC reduced the loss of cardiac function induced by MI, increased wall thickness and prevented the fibrotic scar expansion. After 4 and 12 weeks, the infarct content of CD68+ and CD206+ was similar in control and treated animals. In vitro, we showed that fibrin profoundly influenced the gene expression and the secretome of BMC, simultaneously upregulating both pro- and anti-inflammatory mediators. Furthermore, the conditioned medium from F-BMC significantly increased the proliferation of macrophages in a subsets dependent manner and modulated their gene expression and cytokines secretion. For instance, F-BMC significantly downregulated the expression of Nos2, Il6 and Ccl2/Mcp1 while Arg1, Tgfb and IL10 were upregulated. Interestingly, macrophages educated by F-BMC increased cardiomyoblast proliferation. In conclusion, our study provides evidence that BMC/fibrin-based treatment lowered the infarct extent and improved cardiac function. The macrophage content was unmodified when measured at a chronic stage. Nevertheless, acutely and in vitro, the F-BMC secretome promotes an anti-inflammatory response that stimulates cardiac cell growth. Finally, our study emphases the acute impact of F-BMC educated macrophages on cardiac cell fate.
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