The Role Of S1P2Receptor In Senescence Endothelial Cells

ObjectiveTo determine the role of SIP receptors in the aging process of endothelial cells by exploring the relationship between SIP receptor expression and cellular function of the human umbilical vein endothelial cells (HUVECs), and pulmonary microvascular endothelial cells (PMECs) isolated from rats at different ages. In addition, results from this research will also provide valuable experimental bases for designing preventive means in aging-related cardiovascular diseases.Method1. The expression levels of SIP receptor and endothelial functions during the aging process of HUVECs in vitroIsolated HUVECs were cultured in M199complete medium and sub-cultured every3days. The cells were grouped according to the cumulative population doubling level (CPDL):The young cells (Y) were collected when CPDL was approximately15; the intermediate cells (I) were collected when CPDL was approximately35; and the senescent cells (S) were collected when CPDL was approximately60. The SIP receptor mRNA levels were detectermined by RT-PCR, and the protein levels were estimated by Western blot. The endothelial morphogenesis was assayed in three-dimensional Matrigel. The wound-healing assay was conducted using a scratch-wound model on primary PMEC Monolayers. The chemotaxis of HUVECs was measured by the Transwell migration assay.2. Constructing and screening of the recombinant adenovirus vector with S1P2receptor-targeted shRNA (shRNA-S1P2)Four pairs of complementary single stranded oligonucleotides (ss oligo) were designed and synthesized. Paired ss oligos were denatured and annealed to form a double-stranded oligonucleotide (ds oligo). The ds oligos were cloned into the pRNAT-H1.1/Shuttle vector to form the shuttle plasmid (pRNAT-H1.1/Shuttle-shRNA). After being verified by sequencing, the recombinant plasmids were transfected into HPAEC mediated by liposome. The efficiencies of different pRNAT-H1.1/Shuttle-shRNAs were screened by RT-PCR. The recombinant plasmids were extracted and digested by two restriction enzymes simultaneously (double digestion). The shRNA fragments that induced the most dramatic silencing of S1P2receptor were collected and sub-cloned into an adenoviral expression vector (pAdeno-X). The recombinant plasmid series were identified using restriction enzymes. Linearized pAdeno-shRNA-S1P2was transduced into HEK293A cells. The recombinant adenoviruses were collected and the titer was measured. The effect of S1P2receptor silencing was detected using Western blot.3. The effects of up-and down-regulation of S1P2receptor on the function of HUVECs in vitro:(1) Over-expression of S1P2receptor:The young HUVECs were transfected with either pcDNA3.1/S1P2or the empty vector pcDNA3.1.(2) Silencing of S1P2receptor:The senescent HUVECs were divided into three groups:the blank control group (without transduction), the interfered group (transduction of shRNA-S1P2adenovirus vector) and the interference control group (transduction of control luciferase shRNA-Luc adenovirus vector). The levels of S1P2receptor mRNA and protein after transductions were measured by RT-PCR and Western blot, respectively. The functional assays (the endothelial morphogenesis assay, the wound-healing assay and the chemotaxis) were conducted as same as that in section1.4. The expression levels of SIP receptor and endothelial functions in the pulmonary microvascular endothelial cells (PMECs) isolated from aged rats(1) The PMECs were isolated from young and aged rats. The levels of the SIP receptors mRNA and protein in PMECs were detected by RT-PCR and Western blot, respectively. The functional assays were conducted as same as that in section1.(2) The effects of SIP receptor silencing on the functions of PMECs from the aged rats. The PMECs isolated from the aged rats were divided into three groups:blank control group (without transduction), interfered group (transduction of shRNA-S1P2adenovirus vector) and interference control group (transduction of control shRNA-Luc adenovirus vector). The levels of the SIP receptors mRNA and protein in PMECs after transductions were detected by RT-PCR and Western blot, respectively. The functional assays were conducted as same as that in section1.Results1. Both mRNA and protein levels of S1P2receptor increased significantly in HUVECs during the in vitro senescence, while the abilities of morphogenetic, wound healing and chemotactic responses decreased significantly.2. Over-expression of S1P2receptor in young HUVECs in vitro significantly reduced the ability of morphogenetic, wound healing and chemotactic responses.3. Silencing of S1P2receptor in senescent HUVECs in vitro significantly increased the ability of morphogenetic, wound healing and chemotactic responses.4. The expression of S1P2receptor is significantly increased in PMECs of the aged rats, and the ability of morphogenetic, wound healing and chemotactic responses is significantly decreased. However, silencing of S1P2receptor significantly increased the ability of morphogenetic, wound healing and chemotactic responses.Conclusions1. During the in vitro aging process, the expression of S1P2receptor in the HUVECs is inversely correlated with the abilities of morphogenetic, wound healing and chemotactic responses.2. Results from over-expression and silencing experiments with the HUVECs demonstrated that S1P2receptor is the cause of the functional changes in morphogenetic, wound healing and chemotactic responses in the senescent HUVECs in vitro.3. Elevated levels of S1P2receptor mediates the impairment functions of morphogenetic, wound healing and chemotactic responses in the PMECs from the aged rats.