Rho-kinase inhibitor hydroxyfasudil protects against HIV-1 Tat-induced dysfunction of tight junction and neprilysin/Aß transfer receptor expression in mouse brain microvessels.

HIV-1 transactivator protein (Tat) induces tight junction (TJ) dysfunction and amyloid-beta (Aß) clearance dysfunction, contributing to the development and progression of HIV-1-associated neurocognitive disorder (HAND). The Rho/ROCK signaling pathway has protective effects on neurodegenerative disease. However, the underlying mechanisms of whether Rho/ROCK protects against HIV-1 Tat-caused dysfunction of TJ and neprilysin (NEP)/Aß transfer receptor expression have not been elucidated. C57BL/6 mice were administered sterile saline (i.p., 100 µL) or Rho-kinase inhibitor hydroxyfasudil (HF) (i.p., 10 mg/kg) or HIV-1 Tat (i.v., 100 µg/kg) or HF 30 min before being exposed to HIV-1 Tat once a day for seven consecutive days. Evans Blue (EB) leakage was detected via spectrophotometer and brain slides in mouse brains. The protein and mRNA levels of zonula occludens-1 (ZO-1), occludin, NEP, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in mouse brain microvessels were, respectively, analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Exposure of the mice to HIV-1 Tat increased the amount of EB leakage, EB fluorescence intensity, blood-brain barrier (BBB) permeability, as well as the RAGE protein and mRNA levels, and decreased the protein and mRNA levels of ZO-1, occludin, NEP, and LRP1 in mouse brain microvessels. However, these effects were weakened by Rho-kinase inhibitor HF. Taken together, these results provide information that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-induced dysfunction of TJ and NEP/Aß transfer receptor expression in the C57BL/6 mouse brain. These findings shed some light on potentiality of inhibiting Rho/Rock signaling pathway in handling HAND.

Role of Cav-1 in HIV-1 Tat-Induced Dysfunction of Tight Junctions and Aß-Transferring Proteins.

OBJECTIVE: To evaluate the role of caveolin-1 (Cav-1) in HIV-1 Tat-induced dysfunction of tight junction and amyloid ß-peptide- (Aß-) transferring proteins. METHODS: A Cav-1 shRNA interference target sequence was cloned into the lentiviral vector pHBLV-U6-Scramble-ZsGreen-Puro and verified by double enzyme digestion and DNA sequencing. Human cerebral microvascular endothelium (HBEC-5i) cells were transduced with viral particles made in 293T cells by transfection with lentiviral packaging plasmids. HBEC-5i cells transduced with Cav-1 shRNA or Ctr shRNA were exposed to HIV-1 Tat for 24 h, and the protein and mRNA levels of the tight junction protein occludin, Aß-transferring protein, receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein- (LRP-) 1, and RhoA were evaluated with Western blot and real-time reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively. RESULTS: After sequencing, an RNA interference recombinant lentivirus expressing a vector targeting Cav-1 was successfully established. The recombined lentiviral particles were made by using 293T cells to package the recombined lentiviral vector. A stable monoclonal cell line with strong GFP expression was acquired with a Cav-1 knockdown rate of 85.7%. The occludin protein and mRNA levels in the Ctr shRNA group were decreased with HIV-1 Tat exposure but were upregulated in the Cav-1 shRNA group. The HIV-1 Tat-induced alterations of RAGE and LRP-1 protein and mRNA levels in the Ctr shRNA group were attenuated in the Cav-1 shRNA group. The RhoA protein levels in the Ctr shRNA group were upregulated by HIV-1 Tat exposure but were downregulated in the Cav-1 shRNA group. CONCLUSION: These results show that HIV-1 Tat-induced downregulation of occludin and LRP-1 and upregulation of RAGE and RhoA may result in the accumulation of Aß in the brain. Silencing the Cav-1 gene with shRNA plays a key role in the protection against HIV-1 Tat-induced dysfunction of the blood-brain barrier and Aß accumulation.