Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Autophagy and angiogenesis inhibition

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors

Abstract 1385: Fusion protein containing RGD-endostatin and human Fc of IgG4improves anti-angiogenic and anti-tumor activity

Abstract Inhibiting angiogenesis is a promising strategy for cancer therapy. Endostatin, a C-terminal fragment of collagen XV[[Unsupported Character - &amp;#1064;]], is an endogenous inhibitor of angiogenesis. We have earlier shown that addition of integrin-targeting sequences to a mutant endostain (P125A-endo) improved localization to tumor vasculature. In the present study, we investigated whether genetically fusing endostatin to Fc region of human IgG can prolong circulatory half-life and improve anti-tumor activity. Two types of constructs were made: a) RGD-endostatin-Fc and b) Endostatin-RGD-Fc. The location of the RGD moiety was either placed at the NH2-terminus or between the carboxyl terminus of endostatin and Fc. Both constructs were expressed in HEK-293 cells and purified using affinity chromatography. Our results show the biological activity of endostatin was significantly enhanced by linking to the Fc region of IgG when compared to P125A-endostatin expressed in yeast. Both RGD-Endo-Fc and Endo-RGD-Fc were able to bind endothelial cells very efficiently and inhibited endothelial cells proliferation, migration in vitro and angiogenesis in vivo. We then investigated the ability of the fusion protein to inhibit ovarian cancer growth in a xenograft model. Once a week administration of the fusion protein into mice bearing established ovarian tumors inhibited tumor growth and increased their survival. Anti-tumor activity of the fusion protein was comparable to Bevacizumab-mediated suppression of tumor growth. Furthermore, when mice were treated with a combination of the fusion protein and Bevacizumab, more than additive inhibition of tumor growth was observed. These data suggest that fusion with Fc-fragment can improve the anti-angiogenic and anti-tumor activity of endostatin. Moreover, our results support a strategy to combine two anti-angiogenic reagents such as anti-VEGF antibody and endostatin-Fc fusion protein to additively inhibit ovarian cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1385.</jats:p

Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells

Abstract Inhibition of endothelial cell proliferation and angiogenesis is emerging as an important strategy in cancer therapeutics. Kringle 5 (K5) of human plasminogen is a potent angiogenesis inhibitor. Previous studies have shown K5 exposure promotes caspase activity and apoptosis in endothelial cells. Here we report that K5 treatment evokes an autophagic response in endothelial cells that is specific and initiated even in the absence of nutritional stress. Endothelial cells exposed to K5 up-regulated Beclin 1 levels within a few hours. Furthermore, progressively increasing amounts of antiapoptotic Bcl-2 were found to be complexed with Beclin 1, although total levels of Bcl-2 remained unchanged. Prolonged exposure to K5 ultimately led to apoptosis via mitochondrial membrane depolarization and caspase activation in endothelial cells. Knocking down Beclin 1 levels by RNA interference decreased K5 induced autophagy but accelerated K5-induced apoptosis. These studies suggest that interfering with the autophagic survival response can potentiate the antiangiogenic effects of Kringle 5 in endothelial cells

Inhibition of ovarian cancer by RGD-P125A-endostatin-Fc fusion proteins

Previous studies have shown that a single point mutation in endostatin at position 125 (P125A) can improve the biological activity of endostatin. Addition of an integrin-targeting moiety, R-G-D, resulted in better localization to tumor vasculature and improved the antiangiogenic activity of endostatin. Because endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In our study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its antiangiogenic and antitumor effects in athymic mice. Two genetic constructs were made, RGD-P125A-endostatin-Fc (RE-Fc) and P125A-endostatin-RGD-Fc (ER-Fc). Both constructs were cloned and expressed in mammalian cells. Purified fusion proteins inhibited endothelial cell migration and proliferation better than yeast-derived P125A-endostatin. Both RE-Fc and ER-Fc inhibited ovarian cancer growth and were found to be as effective as Bevacizumab treatment. Fusion protein showed marked increased half-life. Combination treatment with Bevacizumab and ER-Fc showed additive inhibition of ovarian cancer growth. These studies demonstrate that genetic fusion with human IgG4-Fc increases the half-life of P125A-endostatin and can be used along with Bevacizumab to improve antiangiogenic and antitumor activities

Abstract 2020: MiR-210 modulates the hypoxic response in endothelial and ovarian cancer cells

Abstract Tumor growth and progression is intricately linked to hypoxia induced adaptive changes. Hypoxia is known to alter chemosensitivity, upregulation of putative cancer initiating stem cells and the microenvironment. The multitude of these changes is brought about by differential gene expression. Hypoxia inducible transcription factor-1 alpha (HIF-α) is a key transducer of altered gene expression under low oxygen or ischemia. In addition to the transcriptional activation of multiple genes, hypoxia also alters miRNA levels in tumor and vascular endothelial cells. miRNAs are small noncoding RNAs ∼22 nucleotides in length representing 1%-2% of the eukaryotic transcriptome. We used microarrays to determine relative changes in 467 miRNAs in endothelial and MA148 ovarian cancer cells exposed to hypoxia. One of the miRNAs, miR-210 was significantly up-regulated under hypoxia by microarray analysis (2-fold, P &amp;lt;0.01). miR-210 expression changes were validated by RT-qPCR. Exogenous over expression of miR-210 in endothelial cells increased their proliferative capacity and induced angiogenesis. These effects were reversed by morpholino antagomiR specific for miR-210. In-vivo studies using transgenic zebrafish showed that miR-210 can increase angiogenesis which could be readily reversed by morpholino antagomiR. These studied together showed that miR-210 plays an important role in angiognenesis. We then over-expressed miR-210 in ovarian cancer cells. While in vitro growth rate was not altered, over expression of miR-210 changed the kinetics of tumor growth. Taken together, these data implicate an important role for mir-210 in hypoxia induced adaptive changes in tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2020.</jats:p

Abstract 3980: Hypoxia-induced microRNA-424 targets CUL2 to stabilize HIF-α isoforms and promotes angiogenesis

Abstract Chronic hypoxia, a hallmark of many tumors, is associated with angiogenesis and tumor progression. Adaptive changes to oxygen availability are critical for cell survival and homeostasis. Cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for a novel mechanism regulating HIF-1α and HIF-2α levels in endothelial cells during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in microvascular EC, HUVEC and EC progenitor cells purified from the blood. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 in EC was regulated by PU.1-dependent transactivation. PU.1 expression was in turn controlled by RUNX-1 and C/EBPα. Knocking down either C/EBPα or RUNX-1 or PU.1 attenuated hypoxia-driven expression of miR-424 and nuclear translocation of HIF-1α. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by specific morpholinos. These results suggest that miR-424 plays an important physiological role in angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2011-3980</jats:p

Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and beta-catenin levels

Endostatin is a well-characterized endogenous inhibitor of angiogenesis that affects cell proliferation and migration by inhibiting integrin and Wnt-mediated signalling pathways. Here, we show that endothelial cells treated with native and P125A-endostatin activate autophagy. Because autophagy can either be protective or induce programmed cell death, experiments were carried out to understand the signalling pathways leading to autophagy in endothelial cells. P125A-endostatin treatment increased the levels of Beclin 1, a crucial molecule in vesicle nucleation and autophagy. The treatment also reduced the levels of Bcl-2, Bcl-x(L) and beta-catenin; however, progressively increasing amounts of Bcl-2 and Bcl-x(L) were found to be complexed with Beclin 1. Increased beta-catenin and Wnt-mediated signalling reduced Beclin 1 levels and rescued endothelial cells from endostatin-induced autophagy. Finally, knocking down Beclin 1 levels by RNA interference decreased autophagy and accelerated caspase activation in endostatin-treated cells. These studies suggest that endothelial cells may initiate autophagy as a survival response to limit the effects of angiogenesis inhibitors. Thus, interfering with autophagy can potentiate the effects of endostatin by promoting a switch to apoptosis

Adeno-associated virus-mediated delivery of a mutant endostatin in combination with carboplatin treatment inhibits orthotopic growth of ovarian cancer and improves long-term survival

A human ovarian cancer cell line, which migrates to mouse ovaries and establishes peritoneal carcinomatosis, was used to evaluate the cooperative effect of an antiangiogenic gene therapy combined with chemotherapy. The ovarian carcinoma cell line MA148 was genetically modified by "Sleeping Beauty" transposon-mediated delivery of DsRed2 fluorescent protein. Stable, high-level expression of DsRed protein enabled in vivo imaging of peritoneal dissemination of ovarian cancer. Both external and internal imaging, along with histopathology, showed migration of i.p. injected human ovarian cancer cell line to mouse ovaries. Using this model, we evaluated the effect of adeno-associated virus (AAV)-mediated expression of a mutant endostatin either alone or in combination with carboplatin treatment. A single i.m. injection of recombinant AAV (rAAV)-mutant human endostatin with P125A substitution (P125A-endostatin) showed sustained expression of mutant endostatin. Antiangiogenic gene therapy inhibited orthotopic growth of ovarian cancer and resulted in 33% long-term tumor-free survival. A single cycle of carboplatin treatment combined with mutant endostatin gene therapy resulted in 60% of the animals remaining tumor free for >200 days, which was significantly better than rAAV-LacZ and/or carboplatin. Combination treatment delayed tumor appearance in 40% of the animals, wherein the residual tumors were smaller in size with limited or no peritoneal metastasis. These studies suggest that AAV-mediated gene therapy of P125A-endostatin in combination with carboplatin is a useful method to inhibit peritoneal dissemination of ovarian carcinoma