The Cdh5-CreERT2 transgene causes conditional Shb gene deletion in hematopoietic cells with consequences for immune cell responses to tumors

Abstract

The tamoxifen-responsive conditional Cdh5-CreERT2 is commonly used for endothelial cell specific conditional deletion of loxP-flanked gene sequences. To address the role of endothelial cell Shb gene for B16F10 melanoma immune responses, tamoxifen-injected Cdh5-CreERT2 /WT and Cdh5-CreERT2 / Shb flox/flox mice received subcutaneous tumor cell injections. We observed a decrease of tumor myeloid cell Shb mRNA in the tamoxifen treated Cdh5-CreERT2 / Shb flox/flox mice, which was not present when the mice had undergone a preceding bone marrow transplantation using wild type bone marrow. Differences in CD4+/FoxP3+ Tregs were similarly abolished by a preceding bone marrow transplantation. In ROSA26-mTmG mice, Cdh5-CreERT2 caused detectable floxing in certain bone marrow populations and in spleen cells. Floxing in bone marrow could be detected two months after tamoxifen treatment. In the spleen, however, floxing was undetectable two months after tamoxifen treatment, suggesting that Cdh5-CreERT2 is operating in a non-renewable population of hematopoietic cells in this organ. These data suggest that conditional gene deletion in hematopoietic cells is a potential confounder in experiments attempting to assess the role of endothelial specific effects. A cautious approach to achieve an endothelial-specific phenotype would be to adopt a strategy that includes a preceding bone marrow transplantation.

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

“…Future refinement of EC-TRAP taking advantage of intersectional genetic approaches based on combined expression of 2 or more EC markers (68) may lead to greater resolution of EC subsets within a tissue, particularly in combination with single-cell RNAseq methods. This would be particularly valuable as many of the widely used Cre models to target ECs, also target (subsets of) hematopoietic cells, including Tek-Cre as described here, as well as other commonly used EC-specific Cre transgenes (38,69,70). Finally, future applications of this approach should significantly advance our understanding of EC function in vivo under both physiologic and pathophysiologic conditions, potentially leading to the identification of new disease-specific biomarkers as well as potential novel therapeutic targets.…”

c-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly

“…3O-Q). This is pertinent, given the known role of c-Src in osteoclasts, the role of macrophages in regression and the recent data demonstrating Cdh5CreERT2 mice can display non-endothelial Cre activity (He et al, 2019). Taken together, these results suggest that, although c-Src is not required for the formation of the vascular lumen, it is required for the stability of newly formed vessels.…”

A miRNA signature in endothelial cell-derived extracellular vesicles in tumor-bearing mice

“…It is presumed therefore that other sub cellular particles >150 nm will contain both ZSGreen and miRNAs but these particles would have to be purified using different approaches (e.g. differential centrifugation and sucrose gradients); similarly, the quantification of ZSGreen + vesicles using NTA could include co-purified heterogeneous ZSGreen + particles also found in mouse plasma, or particles derived from a small population of Cdh5 + hematopoietic cells, potentially skewing the ratio of total circulating EVs:EC-derived EVs 25 . In our in vitro assays, there also appear to be Lamp1 + endosomes that are ZSGreen − resulting in an underestimation of EC-EVs/exosomes in the circulation and identification of only a partial miRNA signature in EC-EVs.…”