Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

Nankai University School of Medicine

Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for the treatment of degenerative diseases or injury and may provide an alternative to cell-based therapy. However, the compositions in MSC-derived exosomes are highly influenced by the microenvironment in which their original cells reside. Here, we hypothesized that a nitric oxide (NO)-releasing polymer can boost the proangiogenic compositions of exosomes and enhance their proangiogenic capacity. Our results demonstrated that exosomes, released from human placenta-derived MSCs (hP-MSCs) by NO stimulation, augment the angiogenic effects of human umbilical vein endothelial cells (HUVECs) in vitro. Moreover, exosomes released from hP-MSCs by NO stimulation revealed superior angiogenic effects and ameliorated limb function in a murine model of hind limb ischemia. Further analysis demonstrated that increased VEGF and miR-126 levels in exosomes released from hP-MSCs by NO stimulation were identified as a novel mechanism contributing to the increased capacity of these exosomes to promote angiogenic processes. In conclusion, designing specific microenvironments for in vitro stem cell culture, such as those containing bioactive material, will facilitate the development of customized exosomes encapsulating a beneficial composition of stem cells for cell-free therapeutic applications.

Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.

Angiogenesis is critical for the growth of tumor by supplying nutrients and oxygen that exacerbates the metastasis and progression of cancer. Noninvasive imaging of angiogenesis during the tumor therapeutic processes may provide novel opportunities for image-guided tumor management.

Molecular Imaging of Tumor Angiogenesis and Therapeutic Effects with Dual Bioluminescence.

Transplantation of endothelial cells (ECs) holds great promise for treating various kinds of ischemic diseases. However, the major challenge in ECs-based therapy in clinical applications is to provide high quality and enough amounts of cells. In this study, we developed a simple and efficient system to direct endothelial differentiation of mouse embryonic stem cells (ESCs) using a controllable chitosan nitric oxide (NO)-releasing hydrogel (CS-NO). ESCs were plated onto the hydrogel culture system, and the expressions of differentiation markers were measured. We found that the expression of Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) increased obviously under the controlled NO releasing environment. Moreover, the Flk-1 upregulation was accompanied by the activation of the phospho-inositide-3 kinase (PI3K)/Akt signaling. We also found that in the presence of the PI3K inhibitor (LY294002), the endothelial commitment of ESCs was abolished, indicating the importance of Akt phosphorylation in the endothelial differentiation of ESCs. Interestingly, in the absence of NO, the activation of Akt phosphorylation alone by using AKT activator (SC-79) did not profoundly promote the endothelial differentiation of ESCs, suggesting an interdependent relationship between NO and the Akt phosphorylation in driving endothelial fate specification of ESCs. Taken together, we demonstrated that NO releasing in a continuous and controlled manner is a simple and efficient method for directing the endothelial differentiation of ESCs without adding growth factors.

Nitric oxide releasing hydrogel promotes endothelial differentiation of mouse embryonic stem cells.

Mesenchymal stem cell (MSC) transplantation has emerged as a very promising strategy for the treatments of peripheral artery disease (PAD). However, MSC-based therapies are limited by low cell retention and survival rate in the ischemic zone. Small molecular (SM) hydrogels have shown attractive abilities to enhance the therapeutic effects of human MSCs via promoting their proliferation or maintaining their differentiation potential. Here, we designed and synthesized a new bioactive and biocompatible hydrogel, Nap-GFFYK-Thiol, using disulfide bonds as cleavable linkers to control the molecular self-assembly and we hypothesized this hydrogel could enhance the retention and engraftment of human placenta-derived MSCs (hP-MSCs) in a mouse ischemic hindlimb model. In vitro results demonstrated that the Nap-GFFYK-Thiol hydrogel increased cell viability through paracrine effects. Moreover, it enhanced the proangiogenic and anti-apoptotic effects of hP-MSCs. In vivo, Nap-GFFYK-Thiol hydrogel improved the hP-MSC retention in the murine ischemic hindlimb model as visualized by bioluminescence imaging. Furthermore, cotransplantation of hP-MSCs with hydrogel improved blood perfusion, leading to superior limb salvage. These therapeutic effects may attribute to reduced inflammatory cell infiltration, enhanced angiogenesis as well as suppressed collagen deposition. In conclusion, the Nap-GFFYK-Thiol hydrogel fabricated using disulfide bonds as cleavable linkers serves as an artificial niche for promoting hP-MSC survival and proangiogenic factor secretion in PAD therapy and thereby provide an alternative strategy for PAD therapy. STATEMENT OF SIGNIFICANCE: Although several phase I/II clinical trials of MSC-based treatments for critical limb ischemia (CLI) are ongoing, MSC-based therapies are still challenged by the low quality and quantity of cells in the ischemic zone, especially in cases of extensive or irreversible damage. Hydrogels have favorable biocompatibility and safety records in the medical field. In the current study, we engineered a new bioactive and biocompatible hydrogel, Nap-GFFYK-Thiol, using disulfide bonds as cleavable linkers to enhance the therapeutic efficacy of human placenta-derived MSCs (hP-MSCs) in mouse limb ischemia model. Notably, Nap-GFFYK-Thiol hydrogel acts as an artificial niche for promoting hP-MSC survival and proangiogenic factor secretion in PAD therapy, which further promoted the restoration of blood perfusion and regeneration of muscle cells. Considering the proangiogenic effect of Nap-GFFYK-Thiol on hP-MSCs, our results may provide a new strategy for the treatment of PAD.

Self-assembled GFFYK peptide hydrogel enhances the therapeutic efficacy of mesenchymal stem cells in a mouse hindlimb ischemia model.

Extracellular vesicles (EVs) attract much attention in liver pathology because they regulate cell-cell communication and many pathophysiological events by transferring their cargos. Monitoring and understanding the in vivo fate and therapeutic capacity of these EVs is critical for the development and optimization of EV-based diagnosis and therapy. Herein, we demonstrate the use of an aggregation-induced emission luminogen, DPA-SCP, for the real-time tracking of EVs derived from human placenta-derived mesenchymal stem cells (MSCs) and their therapeutic effects in a mouse acute liver injury (ALI) model. In vitro, DPA-SCP does not alter the inherent characteristics of MSC-derived EVs and shows extremely low toxicity. Moreover, DPA-SCP exhibited superior labeling efficiency and tracking capability to the most popular commercial EV trackers, PKH26 and DiI. In vivo, DPA-SCP precisely and quantitatively tracked the behaviors of EVs for 7 days in the mouse ALI model without influencing their regenerative capacity and therapeutic efficacy. The therapeutic effects of EVs may attribute to their ability for reducing inflammatory cell infiltration, enhancing cell survival and antiapoptotic effects. In conclusion, DPA-SCP with an AIE signature serves as a favorable and safe tracker for in vivo real-time imaging of EVs in liver regeneration.

In Vivo Real-Time Imaging of Extracellular Vesicles in Liver Regeneration via Aggregation-Induced Emission Luminogens.

Vascular grafts often exhibit low patency rates in clinical settings due to the pathological environment within the patients requiring the surgery. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have attracted increasing attention. These sEVs contain many potent signaling molecules that play important roles in tissue regeneration, such as microRNA and cytokines. In this study, a sEVs-functionalized vascular graft was developed, and in vivo performance was systematically evaluated in a rat model of hyperlipidemia. Electrospun poly (ε-caprolactone) (PCL) vascular grafts were first modified with heparin, to enhance the anti-thrombogenicity. MSC-derived sEVs were loaded onto the heparinized PCL grafts to obtain functional vascular grafts. As-prepared vascular grafts were implanted to replace a segment of rat abdominal artery (1 cm) for up to 3 months. Results showed that the incorporation of MSC-derived sEVs effectively inhibited thrombosis and calcification, thus enhancing the patency of vascular grafts. Furthermore, regeneration of the endothelium and vascular smooth muscle was markedly enhanced, as attributed to the bioactive molecules within the sEVs, including vascular endothelial growth factor (VEGF), miRNA126, and miRNA145. More importantly, MSC-derived sEVs demonstrated a robust immunomodulatory effect, that is, they induced the transition of macrophages from a pro-inflammatory and atherogenic (M1) phenotype to an anti-inflammatory and anti-osteogenic (M2c) phenotype. This phenotypic switch was confirmed in both in vitro and in vivo analyses. Taken together, these results suggest that fabrication of vascular grafts with immunomodulatory function can provide an effective approach to improve vascular performance and functionality, with translational implication in cardiovascular regenerative medicine.

Kaiyue Zhang

Nankai University School of Medicine

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis