Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks.

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-ß signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury.

Individuals with spinal cord injury (SCI) usually suffer from permanent neurological deficits, while spontaneous recovery and therapeutic efficacy are limited. Here, we demonstrate that when given intranasally, exosomes derived from mesenchymal stem cells (MSC-Exo) could pass the blood brain barrier and migrate to the injured spinal cord area. Furthermore, MSC-Exo loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) could attenuate the expression of PTEN in the injured spinal cord region following intranasal administrations. In addition, the loaded MSC-Exo considerably enhanced axonal growth and neovascularization, while reducing microgliosis and astrogliosis. The intranasal ExoPTEN therapy could also partly improve structural and electrophysiological function and, most importantly, significantly elicited functional recovery in rats with complete SCI. The results imply that intranasal ExoPTEN may be used clinically to promote recovery for SCI individuals.

Mature vessel networks in engineered tissue promote graft-host anastomosis and prevent graft thrombosis.

Graft vascularization remains one of the most critical challenges facing tissue-engineering experts in their attempt to create thick transplantable tissues and organs. In vitro prevascularization of engineered tissues has been suggested to promote rapid anastomosis between the graft and host vasculatures; however, thrombotic events have been reported upon graft implantation. Here, we aimed to determine whether in vitro vessel maturation in transplantable grafts can accelerate vascular integration and graft perfusion and prevent thrombotic events in the grafts. To this end, endothelial cells and fibroblasts were cocultured on 3D scaffolds for 1, 7, or 14 d to form vasculature with different maturation degrees. Monitoring graft-host interactions postimplantation demonstrated that the 14-d in vitro-cultured grafts, bearing more mature and complex vessel networks as indicated by elongated and branched vessel structures, had increased graft-host vessel anastomosis; host vessel penetration into the graft increased approximately eightfold, and graft perfusion increased sixfold. The presence of developed vessel networks prevented clot accumulation in the grafts. Conversely, short-term cultured constructs demonstrated poor vascularization and increased thrombus formation. Elevated expression levels of coagulation factors, von Willebrand factor (vWF), and tissue factor (TF), were demonstrated in constructs bearing less mature vasculature. To conclude, these findings demonstrate the importance of establishing mature and complex vessel networks in engineered tissues before implantation to promote anastomosis with the host and accelerate graft perfusion.

Oscillatory Strain Promotes Vessel Stabilization and Alignment through Fibroblast YAP-Mediated Mechanosensitivity.

Endothelial cells form the interior layer of blood vessels and, as such, are constantly exposed to shear stress and mechanical strain. While the impact of shear stress on angiogenesis is widely studied, the role of mechanical strain is less understood. To this end, endothelial cells and fibroblasts are cocultured under oscillatory strain to create a vessel network. The two cell types show distinctly different sensitivities to the mechanical stimulation. The fibroblasts, sense the stress directly, and respond by increased alignment, proliferation, differentiation, and migration, facilitated by YAP translocation into the nucleus. In contrast, the endothelial cells form aligned vessels by tracking fibroblast alignment. YAP inhibition in constructs under mechanical strain results in vessel destruction whereas less damage is observed in the YAP-inhibited static control. Moreover, the mechanical stimulation enhances vessel development and stabilization. Additionally, vessel orientation is preserved upon implantation into a mouse dorsal window chamber and promotes the invading host vessels to orient in the same manner. This study sheds light on the mechanisms by which mechanical strain affects the development of blood vessels within engineered tissues. This can be further utilized to engineer a more organized and stable vasculature suitable for transplantation of engineered grafts.

Chemotherapy administration to breast cancer patients affects extracellular vesicles thrombogenicity and function.

Breast cancer (BC) is the most prevalent type of malignancy in women. Extracellular vesicles (EVs) are subcellular membrane blebs that include exosomes and microparticles. STUDY AIMS: To elucidate the effects of chemotherapy administration on BC patients' EVs characteristics and their effects on endothelial cells (EC) functions. METHODS: EVs were isolated from the blood samples of 54 BC patients treated by chemotherapy (25 neo-adjuvant, 29 adjuvant) and from 20 healthy women (control group). Blood samples were taken before chemotherapy and on the day of last chemotherapy administration. In some patients, samples were also evaluated 24 hours after chemotherapy treatment. EVs were characterized by cell origin, thrombogenicity and cytokine content. EVs effects on coagulation, migration, apoptosis and proliferation of endothelial cells were assessed as well. RESULTS: Patient characteristics of the two subgroups were similar except for tumor size. Change in EV expression of BC markers, MUC1 and EpCAM, were found in patient subgroups. EC-EVs were significantly higher in both patient subgroups compared to healthy controls. Higher EVs pro-coagulant activity was found at the end of chemotherapy and a significant increase in the ratio between tissue factor (TF) and TF pathway inhibitor was documented after the first 24hours of exposure to doxorubicin treatment. Furthermore, EVs of neo-adjuvant patients obtained before chemotherapy contained more pro-angiogenic proteins, reduced endothelial cells apoptosis and increased their migration compared to EVs obtained at the same timing from adjuvant patients. CONCLUSIONS: EVs may serve as a biomarker for chemotherapy-related thrombogenicity and may indicate vascular damage even before chemotherapy.

Quantifying continuous-flow dielectrophoretic trapping of cells and micro-particles on micro-electrode array.

An interdigitated electrode array embedded within a micro-channel with forced flow is shown to enable dielectrophoretic (DEP) characterization of particles and/or cells based on measurements of their trapping percentage over a continuous frequency range. A simplified model of the trapping percentage, using spatial averaging of the convective and DEP force, linearly correlated it to the effective DEP force (in its positive mode). Thus, the Clausius-Mossotti factor was fitted to the experimental data, yielding effective electrical characteristics of the particles and/or cells. Also, the generated trapping percentage curve response over a continuous range of frequencies facilitates sorting and detection based on differences other than just the cross-over frequencies.