SLIT3–ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo

Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called “pericytic” MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, “non-pericytic” MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications

Japanese encephalitis virus detections in Australia.

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Results of JEV RT-qPCR testing of brain samples.

Results of JEV RT-qPCR testing of brain samples.</p

Primer scheme used for the amplification of JEV 1600bp amplicon sequencing.

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Positively selected sites within the JEV/Australia/NT_Tiwi Islands/2021 genome.

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Maximum clade credibility phylogenetic tree of complete JEV envelope gene sequences, sourced from GenBank.

The phylogeny was reconstructed under a GTR+G+I nucleotide substitution assumption and an uncorrelated relaxed molecular clock model. Genotype V sequences of JEV were excluded from this phylogeny as they were deemed temporal outliers in regression analysis. Posterior probability values of >0.70 are presented adjacent to nodes as indicated by asterisks. The mean time to most recent common ancestor (tMRCA) is presented above major nodes, with error reported as the 95% highest probability density (95% HPD).</p

Maximum likelihood phylogenetic tree of JEV envelope genes available in GenBank.

The TIM2 model with gamma rate heterogeneity was chosen as the most appropriate model by IQ-TREE v.2.0.6. The results from 1000 bootstrap replicates are given on the nodes and the scale represents the number of nucleotide substitutions per site. (TIF)</p

Sequence alignment of primers and probes used for the detection of JEV at PWLM and ACDP.

Primer and probe positions are shown according to the sequence of the JEV/Australia/NT_Tiwi Islands/2021 genome (OM867669). JEV RT-PCR assays used at PWLM comprised primers and probes in alignments 1 and 2 [34,35]. JEV RT-PCR assays used at ACDP comprised primers and probes in alignments 3 and 4 [32,33]. (TIF)</p

Maximum likelihood phylogenetic tree of complete JEV genomes available in GenBank.

The TN model with gamma rate heterogeneity was chosen as the most appropriate model by IQ-TREE v.2.0.6. The results from 1000 bootstrap replicates are given on the nodes and the scale represents the number of nucleotide substitutions per site.</p

Fig 1 -

Map of the Tiwi Islands showing its proximity to mainland Australia and the Northern Territory. The Tiwi Islands comprise Bathurst Island and Melville Island. The locations of major settlements on these islands are shown. Darwin is the capital city of the Northern Territory. The map was created in QGIS version 3.26.2-Buenos Aires (QGIS Development Team (2022). QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org). The base layer of the map used to generate this figure was downloaded from https://www.abs.gov.au/statistics/standards/australian-statistical-geography-standard-asgs-edition-3/jul2021-jun2026/access-and-downloads/digital-boundary-files and is licensed under a Creative Commons Attribution 4.0 International licence.</p