Intraperitoneal delivery leads to efficient gallbladder CAV1 gene transfer in mice.
Since CAV1 is widely distributed in the gallbladder, intestine, and liver tissues (https://www.proteinatlas.org/ENSG00000105974-CAV1/tissue), we evaluated the effect of different routes of delivery and AAV-mediated CAV1 gene transduction in those three tissues. We compared i.p. and i.v. AAV2/8CAV1 delivery in adult mice. Briefly, 1 × 1011 vg per mice of the AAV2/8CAV1 were administered to 8-week-old mice, either by i.v. or i.p. injection. 8-week post-injection, the liver, gallbladder, and ileum tissues were collected to analyze CAV1 expression. No significant differences were observed in the CAV1 expression and the AAV vector genome copy number in the liver of mice between i.v. and i.p. injection (Figs. 1A and B). And we found that i.p. AAV2/8CAV1 delivery increased transduction efficiency in the gallbladder as compared with i.v. injection (Figs. 1A and B). In addition, we confirmed that AAV2/8CAV1 did not transduce in the ileum at high frequency (< 3% CAV1 gene transduction efficiency), which in consistent with prior findings [14]. Western blot further verified these above results (Fig. 1C).
Intraperitoneal administration of AAV2/8CAV1 prevent CGD, regardless changes of biliary CSI.
CAV1 is considered to participate in the regulation of hepatic lipid accumulation and cholesterol metabolism, thus playing an important role in the pathogenesis of diseases related to aberrant triglyceride or cholesterol metabolism[3]. Therefore, we examined the concentration of bile acid, lecithin, and cholesterol in gallbladder bile of chow-fed or LD-fed mice, after injection of AAV2/8CAV1. 8-week LD feeding would reduce the hepatic and gallbladder CAV1 protein levels of control (uninjected) mice (Fig. 1C). Either i.v. or i.p. administration of AAV2/8CAV1 would elevate the hepatic CAV1 expression collected from 8-week LD-fed mice (Fig. 1C). And CAV1 concentration in the gallbladder was higher in i.p. injected mice than in i.v. injected mice and control mice (Fig. 1C). CAV1 overexpression did not alter the feeding behavior of the mice (Fig. 2A). The intestinal expression of niemann-pick disease, type C1-like intracellular cholesterol transporter 1 (NPC1L1) and the fecal contents of cholesterol also were similar between control mice and AAV2/8CAV1-treated mice after LD feeding (Fig. 2B and C), which implicated that CAV1 over-expression did not change oral cholesterol uptake.
CGD results from the imbalance between bile acids, cholesterol, and phospholipids in the gallbladder bile[4, 7]. The canalicular efflux of cholesterol, bile acids, and phospholipids is mediated by ABCG5/G8, bile salt export pump (BSEP), multidrug resistance protein 2 (MRP2) and multidrug resistance protein 2 (MDR2), respectively, which directly regulate bile cholesterol saturation, were similar between control mice and AAV2/8CAV1 administered mice after LD feeding (Fig. 2B). There were no significant differences in the contents of biliary bile acids, cholesterol, phospholipids or biliary CSI between AAV2/8CAV1-treated or control mice (Fig. 2D). However, the CGD prevalence (Fig. 2E) was similar between control (9/9) and i.v. AAV2/8CAV1 treated mice after 8-week LD feeding (11/13), while which was detected as a lower incidence in LD-fed mice with i.p. AAV2/8CAV1 administration (7/13).
AMPK transactivates ABCG5/G8 gene expression to increase biliary cholesterol output.
Our previous work has shown that global CAV1 deficiency would promote CGD via the progression of the dysfunction of hepatic lipid metabolism and the upregulation of liver X receptor (LXR)-ABCG5/G8 signaling [6]. Here we found that the hepatic triglyceride and cholesterol levels of LD-fed mice administered AAV2/8CAV1 via either the i.v. or i.p. route were markedly reduced as compared with control mice (Fig. 3A).
This has borne out in several studies [15–19], and we have proved that CAV1 over-expression by AAV2/8CAV1 treatment prevented LD feeding induced hepatic steatosis and abnormal lipid metabolism by reducing SREBP1c expression via AMPK pathway activation[20] (Fig. 3B). And AMPK also suppressed hepatic de novo cholesterol synthesis by inhibiting hepatic sterol-regulatory-element-binding protein-2 (SREBP2) maturation [20] (Fig. 3B), which may explain why hepatic cholesterol levels decreased but biliary cholesterol levels remained unchanged (Figs. 2D and 3A). And the treatment of compound c (AMPK inhibitor) removed the protective effects of AAV2/8CAV1 treatment on aberrant hepatic lipid metabolism (Figs. 3A and B). Additionally, hepatic principle bile acid synthesis enzyme cytochrome P450 7A1 (CYP7a1) expression was comparable between control mice and AAV2/8CAV1-treated mice after LD feeding (Fig. 2B), although previous study has shown that AMPK limits the conversion of cholesterol to bile acids by suppressing the hepatic expression of the CYP7a1 in human HepG2 cells [21].
According to a large epidemiological work, AMPK may positively affect CGD via the transactivation of ABCG5/G8 [22]. And animal studies have shown that ABCG5/G8 plays a direct role in the process that leads to bile cholesterol supersaturation, which reduces hepatic cholesterol burden [23]. However, the hepatic cholesterol output and ABCG5/G8 expression remained unchanged between AAV2/8CAV1-injected LD-fed mice, with or without compound c treatment (Figs. 2B, 2D). ABCG5/G8 genes have long been known as a direct target of the oxysterol receptors LXR [12]. Thus, four ABCG5/G8-LUC chimeric constructs were produced and transiently transfected into HepG2 cells to delineate the cis-acting regions of the ABCG5/G8 gene that are directly responsible for transcriptional regulation by LXR and AMPK. As predicted, the synthetic LXR ligand T0901317 compound (1 µM) would induce the expression of luciferase from reporters containing the potential LXR site [12] on ABCG5 gene intron 2 (5'-GGATCACTTGAGGTCA-3'; core similarity = 1.0, matrix similarity = 0.985) or on ABCG8 gene intron 3 (5'-GGATCACCTGAGGTCA-3'; core similarity = 1.0, matrix similarity = 0.935) (Fig. 4A), while this two DNA regions showed a reduced ability to respond to T0901317 in a reporter assay in the presence of AMPK activator (0.5 mM metformin). Furthermore, in consistent with previous reports[24], AMPK activation could impede LXR mediated SREBP1c gene transactivation (Fig. 4B).
Next, the luciferase activities of reports containing the 380 bp ABCG5/ABCG8 intergenic region induced by metformin (0.5 mM) in the absence or presence of T0901317 (1 µM) looked equal (Fig. 4C). Additionally, the report on the analysis of the human ABCG5/ABCG8 intergenic region for potential transcription factor binding sites revealed that there are only two regulatory elements, transcription enhancer factor 1 (TEF1) and GATA, that are present in in mouse and human species [12]. The presence of a GATA site (5'-AGATAA-3') is particularly interesting, because it is known to regulate the expression of ABCG5/G8 [25]. Indeed, AMPK could increase the DNA binding activity of GATA binding protein 4 (GATA4) [26]. And we performed a binding site mutagenesis combined with luciferase reporter assays to show that GATA site mutation impeded the role of AMPK on ABCG5/G8 gene transcription (Fig. 4D). These data implied that LXR and AMPK induced luciferase transactivation by acting on different sites of the ABCG5/G8 gene. And AMPK could suppress LXR-dependent ABCG5/G8 gene transcription.
Intraperitoneal AAV2/8CAV1 delivery prevents CGD via the reduction of gallbladder MUC1 expression and the improvement of gallbladder motility.
Unlike i.v. route, i.p. AAV2/8CAV1 administration reduce CGD prevalence in LD-fed mice, although both of them did not affect LD feeding-induced bile cholesterol saturation (Figs. 2D and E). These data proved that bile cholesterol saturation is required but insufficient for CGD. The formation of cholesterol gallstones is also linked to the accumulation of pronucleating mucins in the gallbladder and its hypomotility, which would enhance the process of bile cholesterol nucleation [7]. It has been reported that epithelial mucin-1 (MUC1) could influence the gallbladder motility and the expression of pronucleating MUC5ac [27]. The gel-forming MUC5ac acted as a protective coating to accelerate the appearance of cholesterol monohydrate crystals, whereas mice with epithelial MUC1 deficiency were resistant to CGD due to decreased MUC5ac expression [28]. And only i.p. AAV2/8CAV1 treatment lowered the gallbladder expression of MUC1 and MUC5ac (“MUC1,5ac”) in LD-fed mice, which was attributed to CAV1-associated gallbladder AMPK activation initiating the microRNA-145 (miR145)/MUC1 axis [29, 30] and inhibiting epidermal growth factor receptor (EGFR)/MUC5ac signalling [31] (Fig. 5A and B). And as expected, these effects on “MUC1,5ac” expression were significantly diminished following AMPK inhibition (Figs. 5A and B).
Additionally, i.v. or i.p. AAV2/8CAV1 injection rescued the diminished gallbladder smooth muscle contraction force in response to acetylcholine in LD-fed mice, while only i.p. AAV2/8CAV1 administration having a mild effect on the damaged gallbladder motility in response to cholecystokinin (Fig. 5C). The difference in gallbladder contraction force between acetylcholine and cholecystokinin stimulation could be a result of restored cholinergic receptor muscarinic 3 (CHRM3) expression in the gallbladder of LD-fed mice treated with AAV2/8CAV1 treatment but who retained a down-regulated gallbladder cholecystokinin a receptor (CCKAR) expression (Fig. 5B). And LD feeding or/and AAV2/8CAV1 treatment did not affect gallbladder cholinergic receptor muscarinic 2 (CHRM2) expression (Fig. 5B). Triglyceride-associated lipid stress contributes to muscarinic receptor damage [32], whereas excessive intracellular cholesterol accumulation impairs CCK signalling [7]. AAV2/8CAV1 treatment could reduce gallbladder free fatty acid and triglyceride contents in 8-week LD-fed mice (Fig. 5D), but only i.p. injection of AAV2/8CAV1 would obviously diminish the cholesterol levels in the gallbladder cells (Fig. 5D). However, the cholesterol content of the gallbladder was comparable between control and i.v. AAV2/8CAV1-treated mice, possibly due to the increased gallbladder MUC1 expression of mice in response to LD feeding, who had a role in the gallbladder absorption of cholesterol from bile (Fig. 5A and 5B). Notably, the expression of gallbladder NPC1L1 was similar between LD-fed mice, without or without AAV2/8CAV1 treatment (Fig. 5B). These findings suggested that the primary mechanism by which i.p. AAV2/8CAV1 administration protects against CGD by preventing the hyperproduction of gallbladder MUC1.