Human Aortic Smooth Muscle Cells Promote Arteriole Formation by Coengrafted Endothelial Cells

Collagen-fibronectin gels containing Bcl-2–transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) implanted in the abdominal walls of immunodeficient mice form mature microvessels invested by host-derived smooth muscle cells (SMC) by 8 weeks. We tested the hypothesis that coengraftment of human aortic SMC (HASMC) could accelerate vessel maturation. To prevent SMC-mediated gel contraction, we polymerized the gel within a nonwoven poly(glycolic acid) (PGA) scaffold. Implanted grafts were evaluated at 15, 30, and 60 days. Acellular PGA-supported protein gels elicited a macrophage-rich foreign body reaction and transient host angiogenic response. When transplanted alone, HASMC tightly associated with the fibers of the scaffold and incorporated into the walls of angiogenic mouse microvessels, preventing their regression. When transplanted alone in PGA-supported gels, Bcl-2-HUVEC retained the ability to form microvessels invested by mouse SMC. Interestingly, grafts containing both Bcl-2-HUVEC and HASMC displayed greater numbers of smooth muscle α-actin–expressing cells associated with human EC-lined arteriole-like microvessels at all times examined and showed a significant increase in the number of larger caliber microvessels at 60 days. We conclude that SMC coengraftment can accelerate vessel development by EC and promote arteriolization. This strategy of EC-SMC coengraftment in PGA-supported protein gels may have broader application for perfusing bioengineered tissues

CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation

IntroductionIn epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear.MethodsTo identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn.Results and ConclusionsWe show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in upregulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as downregulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.Proteomic