Alessio Noghero

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. Identifying proteins associated with EPs that regulate host responses to physiological and pathophysiological processes is of critical importance. Here, we report the findings of our study to gain insight into the proteins associated with NVEPs. We used samples from human semen, the rat brain, and the rhesus macaque (RM) brain and blood to assess the physical properties and proteome profiles of NVEPs from these specimens. The results show significant differences in the zeta potential, concentration, and size of NVEPs across different species. We identified 938, 51, and 509 total proteins from NVEPs isolated from rat brain tissues, RM blood, and human seminal plasma, respectively. The species-specific protein networks show distinct biological themes, while the species-conserved protein interactome was identified with six proteins (ALB, CST3, FIBA/FGA, GSTP1, PLMN/PLG, PPIA) associated with NVEPs in all samples. The six NVEP-associated proteins are prone to aggregation and formation of wide, insoluble, unbranched filaments with a cross-beta sheet quaternary structure, such as amyloid fibrils. Protein-to-function analysis indicates that the six identified proteins are linked to the release of dopamine, immune-mediated inflammatory disease, replication of RNA viruses, HIV-HCV co-infection, and inflammation. These interesting findings have created an opportunity to evaluate NVEPs for their potential use as biomarkers of health and disease. Additional in-depth studies are needed to clarify when and how these proteins sustain their physiological role or transition to pathogenic roles.

Extracellular vesicles (EVs) are rapidly gaining recognition as critical mediators of inter-cellular communication during viral infections. To contribute to fill the gap in knowledge regarding the role of EVs in adenovirus infection, we used human adenovirus type 4 of species Mastadenovirus exoticum (HAdV-E4), a prevalent respiratory and ocular pathogen, and characterized the cargo and biological properties of EVs released by HAdV-E4-infected A549 lung epithelial cells at a pre-lytic stage of infection. Using immunocapture-based isolation and multi-omics approaches, we found that infection profoundly alters the EV uploaded proteome and small non-coding RNA repertoire. Mass spectrometry identified 268 proteins unique to EVs purified from infected cells (AdV-EVs), with enrichment in pathways supporting vesicle trafficking and viral protein translation, and importantly also a few virus-encoded proteins. A small RNA transcriptome analysis showed differential uploading in AdV-EVs of various small non-coding RNAs, including snoRNAs, as well as the presence of virus associated RNAs I and II. Notably, AdV-EVs contained viral genomic DNA and could initiate productive infection upon delivery to naïve cells in the absence of detectable viral particles. Our data suggest that EVs released during the HAdV-E4 infection may serve as vehicles for non-lytic viral dissemination and highlight their possible role in intra-host dissemination

Replication of HIV-1 requires the coordinated action of host and viral transcription factors, most critically the viral transactivator Tat and the host nuclear factor κB (NF-κB). This activity is disrupted in infected cells that are cultured with extracellular vesicles (EVs) present in human semen, suggesting that they contain factors that could inform the development of new therapeutics. Here, we explored the contents of semen-derived EVs (SEVs) from uninfected donors and individuals with HIV-1 and identified host proteins that interacted with HIV Tat and the NF-κB subunit p65. Integrative network and pathway enrichment analyses of these complexes revealed associations with an array of biological functions regulating gene expression. Several proteins in SEVs bound to both Tat and NF-κB p65: the scaffolding and cell signaling regulatory protein AKAP9, the G protein signaling regulator ARHGEF28, the epigenetic reader BRD2, the small nuclear RNA processor INTS1, and the transcription elongation inhibitor NELFB. When complexed with p65, NELFB also interacted with HEXIM1, another transcription elongation inhibitor, suggesting that SEVs may inhibit HIV-1 propagation through multiple networks of transcriptional activation and repression. Exploring these data and the underlying mechanisms may inform the development of more effective or more durable therapeutics against HIV.

Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) are each leading causes of mortality and morbidity worldwide, and present additional treatment considerations when they are comorbid (TBI+HS) as a result of competing pathophysiological responses. The current study rigorously quantified injury biomechanics with high precision sensors and examined whether blood-based surrogate markers were altered in general trauma as well as post-neurotrauma. Eighty-nine sexually mature male and female Yucatan swine were subjected to a closed-head TBI+HS (40% of circulating blood volume; n = 68), HS only ( n = 9), or sham trauma ( n = 12). Markers of systemic (e.g., glucose, lactate) and neural functioning were obtained at baseline, and at 35 and 295 min post-trauma. Opposite and approximately twofold differences existed for both magnitude (device > head) and duration (head > device) of quantified injury biomechanics. Circulating levels of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and ubiquitin C-terminal hydrolase L1 (UCH-L1) demonstrated differential sensitivity for both general trauma (HS) and neurotrauma (TBI+HS) relative to shams in a temporally dynamic fashion. GFAP and NfL were both strongly associated with changes in systemic markers during general trauma and exhibited consistent time-dependent changes in individual sham animals. Finally, circulating GFAP was associated with histopathological markers of diffuse axonal injury and blood–brain barrier breach, as well as variations in device kinematics following TBI+HS. Current findings therefore highlight the need to directly quantify injury biomechanics with head mounted sensors and suggest that GFAP, NfL, and UCH-L1 are sensitive to multiple forms of trauma rather than having a single pathological indication (e.g., GFAP = astrogliosis).

In the last several years, accumulating evidence indicates that noncoding RNAs, especially long-noncoding RNAs (lncRNAs) and microRNAs, play essential roles in regulating angiogenesis. However, the contribution of lncRNA-mediated competing-endogenous RNA (ceRNA) activity in the control of capillary sprouting from the pre-existing ones has not been described so far. Here, by exploiting the transcriptomic profile of VEGF-A-activated endothelial cells in a consolidate three-dimensional culture system, we identified a list of lncRNAs whose expression was modified during the sprouting process. By crossing the lncRNAs with a higher expression level and the highest fold change value between unstimulated and VEGF-A-stimulated endothelial cells, we identified the unknown LINC02802 as the best candidate to take part in sprouting regulation. LINC02802 was upregulated after VEGF-A stimulation and its knockdown resulted in a significant reduction in sprouting activity. Mechanistically, we demonstrated that LINC02802 acts as a ceRNA in the post-transcriptional regulation of Mastermind-like-3 (MAML3) gene expression through a competitive binding with miR-486-5p. Taken together, these results suggest that LINC02802 plays a critical role in preventing the miR-486-5p anti-angiogenic effect and that this inhibitory effect results from the reduction in MAML3 expression.

Angiogenesis requires the temporal coordination of the proliferation and the migration of endothelial cells. Here, we investigated the regulatory role of microRNAs (miRNAs) in harmonizing angiogenesis processes in a three-dimensional in vitro model. We described a microRNA network which contributes to the observed down- and upregulation of proliferative and migratory genes, respectively. Global analysis of miRNA–target gene interactions identified two sub-network modules, the first organized in upregulated miRNAs connected with downregulated target genes and the second with opposite features. miR-424–5p and miR-29a-3p were selected for the network validation. Gain- and loss-of-function approaches targeting these microRNAs impaired angiogenesis, suggesting that these modules are instrumental to the temporal coordination of endothelial migration and proliferation. Interestingly, miR-29a-3p and its targets belong to a selective biomarker that is able to identify colorectal cancer patients who are responding to anti-angiogenic treatments. Our results provide a view of higher-order interactions in angiogenesis that has potential to provide diagnostic and therapeutic insights.

Interferon-α (IFN-α) comprises a family of 13 cytokines involved in the modulation of antiviral, immune, and anticancer responses by orchestrating a complex transcriptional network. The activation of IFN-α signaling pathway in endothelial cells results in decreased proliferation and migration, ultimately leading to suppression of angiogenesis. In this study, we knocked-down the expression of seven established or candidate modulators of IFN-α response in endothelial cells to reconstruct a gene regulatory network and to investigate the antiangiogenic activity of IFN-α. This genetic perturbation approach, along with the analysis of interferon-induced gene expression dynamics, highlighted a complex and highly interconnected network, in which the angiostatic chemokine C-X-C Motif Chemokine Ligand 10 (CXCL10) was a central node targeted by multiple modulators. IFN-α-induced secretion of CXCL10 protein by endothelial cells was blunted by the silencing of Signal Transducer and Activator of Transcription 1 (STAT1) and of Interferon Regulatory Factor 1 (IRF1) and it was exacerbated by the silencing of Ubiquitin Specific Peptidase 18 (USP18). In vitro sprouting assay, which mimics in vivo angiogenesis, confirmed STAT1 as a positive modulator and USP18 as a negative modulator of IFN-α-mediated sprouting suppression. Our data reveal an unprecedented physiological regulation of angiogenesis in endothelial cells through a tonic IFN-α signaling, whose enhancement could represent a viable strategy to suppress tumor neoangiogenesis.

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Neuronal pentraxins (NPTX) and their corresponding receptors (NPTXR) have been studied as synapse-associated proteins in the nervous system, but their role in cancer is largely unknown. By applying a multidisciplinary, high-throughput proteomic approach, we have recently identified a peptide ligand motif for targeted drug delivery to neuroblastoma. Here, we report the sequence similarity between this peptide and a conserved portion of the pentraxin domain that is involved in the homo- and hetero-oligomerization of NPTX2 and NPTXR. We show that, in comparison with normal tissues, NPTX2 and NPTXR are overexpressed in vivo in mouse models, as well as in human Schwannian stroma-poor, stage IV neuroblastoma. Both proteins are concentrated in the vicinity of tumor blood vessels, with NPTXR also present on neuroblastic tumor cells. In vivo targeting of NPTX2 and NPTXR with the selected peptide or with specific antibodies reduces tumor burden in orthotopic mouse models of human neuroblastoma. In vitro interference with this ligand/receptor system inhibits the organization of neuroblastoma cells in tumor-like masses in close contact with vascular cells, as well as their adhesion to normal microenvironment-derived cells, suggesting a role in the cross-talk between tumor and normal cells in the early steps of neuroblastoma development. Finally, we show that NPTX2 is a marker of poor prognosis for neuroblastoma patients. Cancer Res; 75(20); 4265–71. ©2015 AACR.