Generation of Plvap-CreER and Car4-CreER for genetic targeting of distinct lung capillary populations

Pulmonary endothelial cells (ECs) are a vital element surrounding each alveolus, charge of the gas exchange machinery of the lung alveolus. Two recent studies using single-cell RNA sequencing (scRNA-seq) reveals the heterogeneity of the alveolar capillary endothelium, which consists two distinct intermingled alveolar capillary cell types (Gillich et al., 2020; Vila Ellis et al., 2020). One is termed as aerocytes (aCap), and another is termed as general capillary (gCap). aCap expresses high level of carbonic anhydrase IV (CAR4), which is related to the function of gas exchange. gCap expresses high level of plasmalemmal vesicle-associated protein (PLVAP) and appears to be the progenitor cells for capillary homeostasis and repair. Despite this, the extent and function of lung EC heterogeneity remain incompletely understood, due to lack of genetic tools available to genetically target these two capillaries specifically in vivo.

PLVAP is a critical integral membrane glycoprotein component of the endothelial stomatal and fenestral diaphragms (Stan et al., 1999; Stan et al., 2001). PLVAP localizes to diaphragms of fenestrae, caveolae, and transendothelial channels, endowed with a crucial role to form diaphragms. Overexpression or reduction of PLVAP can mediate the formation of caveolar stomatal diaphragms (Stan et al., 2004). The lack of endothelial diaphragms in fenestrae and caveolae of mutant PLVAP-deficient mice leads to defects in the vascular wall of subcutaneous capillaries and decreased survival (Herrnberger et al., 2012). Besides the regulation of vascular permeability, PLVAP is capable of facilitating leukocyte trafficking. Additionally, an evident inhibition of lymphocyte transmigration through the endothelial cells can be observed in the situation of PLVAP blockage (Keuschnigg et al., 2009). In PLVAP-deficient mice, both the delivery of antigens and the transmigration of lymphocytes are amplified (Rantakari et al., 2015). Due to the connection between PLVAP with some diseases (Madden et al., 2004; Carson-Walter et al., 2005; Yamamoto et al., 2007; Yokomori et al., 2021), PLVAP can be a potential novel therapeutic target.

CAR4 is a membrane-associated enzyme, localized on apical surfaces of renal tubular epithelium and endothelium of specialized capillary beds (Tamai et al., 1996). Although CAR4 has been demonstrated to play a role in mediating bicarbonate ions and carbon dioxide transport in many organs like lung and kidney, the function of CAR4 seems not to be essential (Waheed and Sly, 2014). Accompanied by the development of lung, pulmonary expression of CAR4 is dynamically regulated. Meanwhile, selective expression of CAR4 is localized to the luminal side of alveolar capillary endothelial cells (Whitney and Briggle, 1982; Fleming et al., 1993; Niethamer et al., 2020).

Understanding the in vivo functions of two subsets of alveolar capillary endothelium in physiological and pathological conditions requires specific genetic perturbation and manipulation of capillary populations. Taking advantage of Plvap and Car4 as specific markers for gCap and aCap respectively, we genetically engineered animal models to enable Cre recombinase expression in PLVAP-expressing cells and CAR4-expressing cells in an inducible manner. In this study, we developed Plvap-CreER and Car4-CreER knock-in mouse lines, and characterized their recombination efficiency and specificity in mouse tissues.

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