Development of an endothelial cell-restricted transgenic reporter rat: a resource for physiological studies of vascular biology

Abstract

Here, we report the generation of a Cre-recombinase transgenic rat, where iCre is driven using a CDH5 promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated approximately 60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelial-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that on average more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 months), middle (14-16.5 months), and old age (17-19.5 months) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared to that seen in the young rats. Thus, this endothelial restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments and it provides exceptional resolution of endothelium within organ microcirculations for application to translational disease models.

Cytotoxic tau released from lung microvascular endothelial cells upon infection with Pseudomonas aeruginosa promotes neuronal tauopathy

“…As seen in Figure 2E, the lung parenchyma ex vivo expresses tau-eGFP, consistent with abundant tauexpressing cells. The eGFP-tau signal colocalized with tomato lectin, an indicator of the endothelium, and was particularly abundant in capillaries (Figure 2F and G) (30). Images obtained from brain slices also revealed extensive tau-eGFP expression in cells, consistent with the expected neuronal tau expression (data not shown).…”

“…Lung preparation and fluorescence imaging. We used the agarose/gelatin-infused lung slice technique to expand air sacs and vessels to evaluate eGFP expression, tomato lectin Dylight594 (Vector Laboratories) vascular staining, and nuclear staining (DAPI for live cell, Invitrogen), as we have previously described (30). Mice were deeply sedated with an injection of ketamine/xylazine mixture.…”

Pneumonia initiates a tauopathy

“…Gelatin was introduced into the circulation and agarose was introduced into the airway, as described previously. 23 The lung was then cut into 300 µm sections and imaged by confocal microscopy. 23 …”

“…#DL‐1178) and NucBlue (Hoechst nuclear dye; Invitrogen) were infused into the circulation. Gelatin was introduced into the circulation and agarose was introduced into the airway, as described previously 23 . The lung was then cut into 300 µm sections and imaged by confocal microscopy 23 …”

Disrupted endothelial cell heterogeneity and network organization impair vascular function in prediabetic obesity

“…While widely acknowledged to exist (e.g. [34,65,[66][67][68][69]), the functional significance of endothelial cell heterogeneity is only beginning to emerge. In some tissues, heterogeneous cell populations organize into spatial domains with coherent gene expression [70], and the resulting multicellular groupings form local microcircuits, the building blocks of information processing.…”