| Background and ObjectiveDifferent from skeletal or cardiac muscle cells which are terminally differentiated, VSMCs of adult animals retain the high degree of plasticity and can generate reversible phenotypic modulation when they are injuried. Phenotypic modulation of the VSMCs plays a key role in a number of major diseases in humans. In systemic arteries, atherosclerosis, restenosis, pulmonary hypertension and leiomyogenic tumorigenicity are the most widely acknowledged human diseases that are believed to involve VSMCs phenotypic modulation. Phenotypic modulation of pulmonary artery smooth muscle cells can be triggered by hypoxia and related to many signaling molecules, recent research showed that Ang II relate to hypoxia-induced proliferation of cultured pulmonary artery smooth muscle cells.Ang II, an octapeptide hormone, elicits its biological actions by binding to specific membrane-bound receptors on target cells to activate multiple intracellular transduction pathways. Two major Ang II receptor subtypes were identified AT1R and AT2R, and both receptor subtypes belong to the seven transmembrane spanning receptor superfamily coupling to heterotrimeric G proteins. Stimulation of AT1R by Ang II activates the mitogen-activated protein kinases, such as extracellular regulated kinases, as well as the JAK/STAT pathways. AT1R may play an important role in vascular remodeling and phenotypic modulation of VSMCs. Hypoxia contributes to disposition of AT1R on cardiac myocytes and VSMCs.It is determinate for its function that AT1R transport intracellular. After being synthesized, folded, and assembled in the ER, AT1R is transported to the Golgi apparatus and is post-translational modified there. Afterward, it is transported to the plasma membrane. Up to now, few studies on the export of AT1R from the ER through the Golgi apparatus to the cell surface and regulation of receptor function by these processes were investigated.Rab proteins, which are members of the Ras superfamily of monomeric GTPases,deal with intracellular protein trafficking between organelles. Today, Rab GTPases are defined as small GTP-binding proteins belonging to the Ras superfamily, over 60 proteins that are defined by their similar structure, function, and guanine nucleotide binding properties. The Rabs play essential roles in various aspects of vesicular membrane transport on both the exo- and endocytic pathways, and like other members of the Ras superfamily, function as molecular switches through changes in its guanine nucleotide binding status. In its active, GTP-bound form, Rabs could mediate vesicular transport by allowing transport carriers orvesicles to engage specific effectors. It has been known that Rab1 is localized in the ER and Golgi and regulates antegrade protein transport specifically from the ER to the Golgi and between the Golgi compartments. It is known that Rab1 regulates the transport of AT1R from the ER to the cell surface of epithelial cell and cardiocytes. These suggest that we can regulate phenotypic modulation and other activity of PASMCs through altering Rab1 function which can affect the cell-surface expression of AT1R subsequently.The aim of our study was to assess whether Rab1 involve ER-to-Golgi transport which can affect the cell-surface expression and function of AT1R in the RPASMCs under hypoxia, the effect of Rab1 mediated alteration of phenotypic modulation and other activity of RPASMCs was also investigated.Methods:1. RPASMCs were cultured by the tissue explants adhere method from intrapulmonary arteries. The identity of RPASMCs was verified by their typical morphological pattern and by immunofluorescence using specific antibody againstα-SMA, the cells purification was performed by differential adhesion.2. The cell-surface expression of AT1R in RPASMCs was measured by intact cell ligand binding after RPASMCs were exposed to hypoxia and infected with wild-type Rab1 lentivirus or gene-silence Rab1 siRNA lentivirus.3. Laser scanning confocal microscope is used to investigate the cell-surface and subcellular distribution of AT1R in RPASMCs after being infected with Rab1WT lentivirus and Rab1 siRNA lentivirus.4. The expression of VSMC differentiation marker (α-SMA and VIM) and activation of STAT3 was determined by western blot analysis using specific antibodies after the RPASMCs were treated.5. The viability of RPASMCs was determined by MTS assay. The treated cells were plated in a 96-well plate which CellTiter 96? AQueous One Solution Cell Proliferation Assay was added into each well. Absorbance was measured at 490 nm directly from 96-well assay plates with a microplate reader.Results:1. Under phase-contrast microscopy, normal primary RPASMCs had a fusiform-like structure and were grouped together in peak-valley formations. Using immunofluorescence, positive expression ofα-SMA antigen in RPASMCs was demonstrated by the presence of lines in the cytoplasm.2. The mean values of specific [3H] Ang II binding were 556±61 and 725±83 cpm (n = 3 duplicate sets) for the RPASMCs in the normoxic group and those subjected to hypoxia for 48 h, respectively; the mean Ang II binding of AT1R were 550±69, 804±130, and 301±46 cpm from the cells infected with control lentivirus, Rab1WT lentivirus, or Rab1 siRNA lentivirus, respectively; the mean Ang II binding of AT1R were 738±98 and 371±68 cpm from the cells infected with Rab1WT lentivirus, and Rab1 siRNA lentivirus (all cells were subjected to hypoxia)(P<0.01).3. GFP-AT1R was localized at the surface in RPASMCs infected with control lentivirus, in contrast, GFP-AT1R accumulated in the perinuclear regions, co-localized with the ER marker, pDsRed2, in cells infected encoding Rab1 siRNA lentiviral.4. The levels of STAT3 tyrosine phosphorylation increased by 5.37 fold in cells being exposed to hypoxia plus AngII. In contrast, STAT3 phosphorylation in RPASMCs treated with Rab1 siRNA only increased by 1.14 under AngII and ZD7155 (thespecific AT1R antagonist) conditions (P<0.01).5. VSMC marker proteins of RPASMCs, includingα-SMA and VIM, were downregulated after hypoxic exposure plus AngII. However, the expression of the SMC marker proteins in RPASMCs infected with lentiviruses encoding Rab1 siRNA under AngII and ZD7155 conditions was not downregulated so much (P<0.05) .6. Exposure to hypoxia for 48 hours plus AngII resulted in a significant, 159% increase of the OD in comparison with normoxic controls. In contrast, the OD of RPASMCs infected with lentivirus encoding Rab1-siRNA under AngII and ZD7155 conditions, only increased by 27% when compared with that of the normoxic controls, which was a statistically significant difference (P<0.01).Conclusion:1. These data indicate that Rab1 can affect its function by modulating vesicular trafficking of AT1R in pulmonary artery smooth muscle cells.2. Rab1 involve in JAK/STAT signal transduction by adjust the transport of AT1R.3. Rab1 can regulate pulmonary artery smooth muscle cells from differentiated phenotype to dedifferentiated phenotype by mediating the transport of AT1R under hypoxia.4. Rab1 is concerned with activity of pulmonary artery smooth muscle cells.Therefore, Rab1 can be as a target of therapeutic intervention to recover pulmonary vascular remodeling. |
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