CAT-1 As A Novel Cell Adhesion Molecular Integrate The Roles Of Endothelial Integrity And L-arginine Transport

Background:A variety of cardiovascular and cerebrovascular diseases, such as coronary heart disease, hypertension, stroke, pulmonary hypertension is a major killer of human health. Finding effective interventions to these diseases are urgently needs for the people’s livelihood. The etiology of this group of diseases are complex, mostly caused by genetic and environmental and other factors, but the integrity of endothelial damage and endothelial dysfunction is an early key to this group of diseases.Interendothelial junctions play an important role in the maintenance of endothelial integrity and in the regulation of endothelial functions. Intercellular junctions in multicellular organisms are essential for various cellular functions, including morphogenesis, differentiation, proliferation, apoptosis, and migration. Three types of intercellular junctions, namely Gap Junctions (GJs), tight junctions (TJs), and adherens junctions (AJs) have been reported in endothelial cells. Whereas GJs form transmembrane channels between contiguous cells, TJs and AJs form pericellular zipperlike structures along the cell border through their transmembrane homophilic adhesion. Of them, adherens junction is a major structure of the interendothelial junction, and cadherins and nectins are the known transmembrane adhesion proteins.The first member of CAT-1 (mCAT-1, for mouse CAT-1) was originally cloned and identified as the receptor for moloney murine leukemia virus (MMLV). Amino acid similarities observed between the MMLV receptor and L-histidine and L-Arg permeases from Saccaromyces cerevisiae led to the discovery of its physiological function as the Na+-independent transporter of cationic amino acids. L-Arg is the exclusive precursor of NO production in endothelial cells (ECs), nitric oxide synthase (NOS)-mediated NO formation is dependent upon an adequate and continuing supply of L-Arg. However, the intracellular L-Arg content is derived primarily from plasma membrane-dependent transport of extracellular L-Arg. Although L-Arg transport can be mediated by several independent transport activities in mammalian cells, CAT-1 is the predominant mediator of L-Arg transport in ECs. NO is a multifunctional bioactive molecules, it play an important role in maintaining the physiological functions of endothelium through the regulation of vascular smooth muscle tone, inhibition of leukocyte and platelet adhesion to vascular endothelium, inhibition of proliferation and migration of vascular smooth muscle cells. Also, NO is an important signaling molecule in regulating various vasomediators responsible for vascular permeability.Recent studies have shown that CAT-1 plays an important role in the cardiovascular system diseases, for example, overexpression of CAT-1 can alleviate endothelial oxidative stress and high blood pressure caused by obesity. But, the role of CAT-1 on endothelial permeablity remains unclear. Since CAT-1 drives endothelial NO generation and excessive NO increases vascular permeability, we originally hypothesized that CAT-1 upregulation will augment endothelial permeability due to the increased NO generation. It is somewhat unexpected however that CAT-1 overexpression reduced rather than increased the endothelial permeability despite higher NO production as presented in our study, while reduction of CAT-1 expression can increase the endothelial permeability. This intriguing finding led us to investigate whether CAT-1 itself via a NO-independent mechanism, such as strengthening intercellular junctions, regulates endothelial integrity. Therefore, in this subject, we used a CAT-1/GFP fusion construct to explore the mechanism for maintaining the endothelial permeablity of CAT-1.Aims:1、Constructing recombinant vectors harboring CAT-1/GFP, and to verify whether the functional property of recombinant adenovector construct is consistent to native CAT-1;2、To assay whether CAT-1 protein through stablilizing cell-cell junctions maintaining the endothelial monolayer permeablity;3、To verify whether CAT-1 protein possess the biochemical and functional characteristics as cell adhesion molecule;4、To investigate the influences of L-Arg on amino acid transport and cell junction function of CAT-1.Methods:Part 1:1、CAT-1 cDNA containing the full-length CDS was obtained using one-step RT-PCR amplification, after subcloning cDNA into pENTRlA/GFP, LR recombination between pENTR1A-CAT-1/GFP and adenovector as well as lentivector was performed, the resulting adenovirus and lentivirus expressing CAT-1/GFP were finally generated according to the standard protocols and used to infect PAEC and CHO-S cells;2、After CAT-1 adenovirus infecting PAEC, to detect L-Arg transport and NO generation by L-[3H]Arg isotope method, and evaluate whether CAT-1/GFP fusion protein is consistent with native CAT-1 protein;3、PAECs grown on coverslips were observed using laser scanning confocal microscopy at 48h after the infection with the adenovirus harboring CAT-1/GFP, to identify the expression and subcellular localization of CAT-1;4、To detect CAT-1/GFP monomer-dimer form by western blot, after treatment of suspension CHO-S cells with protein cross-linking agent BS3, then detect whether the dimer form of CAT-1/GFP is trans or cis;5、Select the CHO-S cells stably expression of CAT-1/GFP with blasticidin antibiotic, and then to identify whether CAT-1 protein functions as cell adhesion molecule through suspension cell aggregation experiments;Part 2:1、To detect the role of CAT-1 on monolayer endothelial cell permeability using Evans blue (EB)-albumin as a tracer of albumin leak;2、After treatment of endothelial cells with L-Arg, to analyse CAT-1 mediated cell junction assembly/disassembly and subcellular localization of CAT-1 using live-cell confocal microscope;Results:Part 1:1、Restriction enzyme digestion and sequencing analysis confirmed that CAT-1 cDNA sequences and CAT-1/GFP fusion gene expression construct were correct;2、CAT-1/GFP fusion protein possess the functional property of the native CAT-1 Successfully isolation and culture of PAEC cells, and the purity is>95% based on the expression of Ⅷ-R Ag; Ad-CAT-1/GFP infected PAECs accumulated>9-fold L-Arg in a Na+-independent manner (p<0.01) and generated>5-fold NO than blank ad-GFP infected PAEC cells (p<0.01);3、CAT-1 protein localize at sites of interendothelial junctions Unlike control GFP, which distributes evenly in the cytoplasm and nucleus, CAT-1/GFP protein mainly localize at the intercellular junctions with some accumulated in the perinuclear area (newly synthesized/Golgi-localized transporter) after the infection. The accumulation peaked at 48-72h and maintained for at least 7 days after infection; Besides, immunofluorescence staining find that native CAT-1 is also localized at cell-cell junctions.4、trans-homo-interaction of the CAT-1 protein in PAECExperiments were carried out with 4-20% SDS-PAGE, compared with the GFP protein sample, when CAT-1/GFP was boiled for 5 min in the presence of loading buffer with 2% SDS, a 95-kDa band and a 190-kDa band were observed, the higher molecular weight band observed appears to be a CAT-1/GFP homodimer that is stable in the presence of the detergent; A single-cell suspension of CHO-s cells stably expressing CAT-1/GFP was incubated in the presence or absence of bis (sulfosuccinimidyl) suberate protein crosslinker (BS3), the cross-linking of the cell line did not result in the formation of additional bands with molecular masses of about 190kDa that corresponded to the dimeric form, it suggests that dimers appearing resulted from the trans-homo-interaction.5、CAT-1 as a cell adhesion molecule was further demonstrated by cell aggregation experiments CAT-1/GFP and GFP stably expressed CHO-S cells was selected with blasticidin antibiotic, after incubation, CAT-1/GFP expressed cells formed cell aggregates more readily than GFP-expressed cells in Ca2+/Mg2+ plus HBSS (P<0.05).Part 2:1、Reduction of CAT-1 expression in PAEC cells leading to increased endothelial permeabilityThe effect of CAT-1 on the permeability of endothelium was characterized by the clearance rate of Evans Blue-labeled albumin, knockdown of CAT-1 with sh-CAT-1 led to an increase in the clearance rate of Evans Blue-labeled albumin by 26%(±6%, P<0.05) compared with cells transfected with scrambled shRNA; While overexpression of CAT-1/GFP led to a slight decrease in the clearance rate of Evans Blue-labeled albumin but was not significantly different from those transfected with Ad-GFP. This is likely the explanation that baseline CAT-1 expression is sufficient to protect the endothelial permeability, thus increase of CAT-1 expression will not significantly reduce the permeability.2、Extracellular L-Arg exposure abates the cell junction disassembly and decrease the internalization of CAT-1we chose 1mM L-Arg, treatment 15 min for PAEC. Compared to GFP control cells, L-Arg exposure inhibited the cell membrane CAT-1 internalization and retarded cell junction disassembly. Besides, CAT-1 protein can shift from cell junctions to cell free surface upon L-Arg exposue. These results provide experimental evidence for explaining the phenomenon of trans-stimulation and L-Arg paradox.Conclusions1. CAT-1/GFP fusion protein possess the functional property of the native CAT-1, so we can use GFP antibody and green fluorescence to detect the function and subcellular location of CAT-1 protein;2. CAT-1 protein is a nove cell adhesion molecule, and maintaining the permeablity of endothelial monolayer through stabilizing cell-cell junctions;3. L-Arg can stabilizing the CAT-1 mediated cell-cell junctions through inhibiting the internalization of CAT-1;4. L-Arg inhibites the internalization of CAT-1 and promotes the expression of CAT-1 on cell free face, which provide another explanation for trans-stimulation and L-Arg paradox.