1. The Role Of Reactive Oxygen Species In The Differentiation Of Multipotent Vascular Stem Cells Into Vascular Smooth Muscle Cells In Rats. 2. Irisin Promotes Human Umbilical Vein Endothelial Cell Proliferation Through The ERK Signaling Pathway And Partly

Background:Abnormal growth of vascular smooth muscle cells (VSMCs) is a major contributor to cardiovascular disease (CVD), the leading cause of death and disability in the world. Extensive studies of VSMC biology have indicated mature VSMCs to be quiescent and contractile in normal vasculature; however, in response to injury, another unique type of VSMCs (i.e., synthetic VSMCs) becomes apparent. These cells expand in number rapidly and release diverse cytokines and growth factors resulting in a vascular lesion. However, a fundamental question regarding the source of synthetic VSMCs remains unanswered.A widely accepted view is that, in response to vascular injury, the proliferative and synthetic VSMCs are derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs in the tunica media. Other documented sources of synthetic VSMCs include bone marrow-derived stem cells and resident vascular stem cells. However, it is clear that the differentiation of bone marrow-derived stem cells into VSMCs is an exceedingly rare event and the contribution of bone marrow-derived cells to the cellular compartment of the vascular lesion is limited to the transient inflammatory response phase. Furthermore, synthetic VSMCs in a vascular lesion are exclusively derived from within the local vessel wall. Therefore, synthetic VSMCs are likely derived from the dedifferentiation of mature VSMCs and/or resident vascular stem cells. However, the relative contributions of dedifferentiation of mature VSMCs and vascular resident stem cells to the generation of the synthetic VSMCs in the vasculature remain unknown. Intriguingly, a recent report indicated the existence of a small population (<10%) of smooth muscle-myosin heavy chain (SM-MHC) negative (-) stem cells, named media-derived multipotent vascular stem cells (MVSCs), in the media of mature blood vessels. It is the MVSC activation and differentiation, instead of mature VSMC dedifferentiation that results in the formation of synthetic VSMCs that contribute to vascular lesion formation. While this conclusion has been weakened by the lack of information regarding the ultimate tracking fate of VSMCs via conditional SMC lineage tracing, these results reemphasize the importance of definitive characterization of the origins of SMC cultures and urgent need for complete SMC lineage tracing studies in vivo in diverse pathological settings.Objective:The aim of this study was to investigate whether the conventional rat aortic SMC (RASMC) culture is a process involving the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs, as well as the potential mechanism for controlling the synthetic phenotype of RASMCs.Materials and methods:1. Cell culture:RASMCs were enzymatically isolated and cultured in both a regular growth medium (RGM) and a stem cell growth medium (SCGM).2. Cell differentiation:RASMCs maintained in SCGM were differentiated into SMCs by PDGF-BB. Directed differentiation of osteoblasts, adipocytes, and chondrocytes were accomplished by specific induction factors.3. Gene knockdown:Pla2g7 and NEDD8 knockdown were achieved by Pla2g7 and NEDD8 RNA interference (RNAi) approachs.4. Real-time RT-PCR:Gene expression were detected by Real-time RT-PCR.5. Western blot analysis:Protein expression were detected by Western blot.6. Immunofluorescent staining:Protein expression were detected by Immunofluor-escent staining.7. Flow cytometry:Protein expression were detected by Flow cytometry.8. [3H]Thymidine uptake:DNA synthesis was measured by [3H]thymidine uptake and normalized by the number of cells in each group.9. ROS measurement:Intracellular ROS levels were measured with DCHF, which could be rapidly oxidized into the highly fluorescent DCF in the presence of intracellular ROS. Images were randomly chosen to photograph and integrated optical density (IOD) of the images was quantified with Image Pro Plus software.10. Statistics:All experiments were repeated at least three times. Results are shown as means±SD. Group comparisons were performed by one-way analysis of variance (ANOVA). The Holm’s t-test was used for the analysis of differences between different groups. Differences were considered significant at P<0.05.Results:1. Cultured RASMCs express MVSC markers and rapidly differentiate into synthetic VSMCs in a conventional regular culture growth medium (RGM) but not in a stem cell growth medium (SCGM).(1) Regardless of culture conditions, only a very small portion of freshly isolated RASMCs attached and survived. Compared with mature SMCs, the surviving cells express much lower levels of SMC marker genes (aSMA, SM22a, hl-calponin and smoothelin).(2) RGM-cultured cells underwent a process of synthetic SMC differentiation.(3) The mRNA of MVSC markers (NFM, Sox 10 and S100β) in SCGM cultured RASMCs were first upregulated at early passage and thereafter declined, finally returning to their basal level. In contrast, the mRNA of these MSVC markers did not change in RASMCs cultured in RGM over time, relative to the freshly isolated cells.(4) Immunofluorescent staining revealed that almost 100% of SCGM-cultured cells at P1 expressed MVSC markers.Western blot and FACS analysis revealed that RASMCs cultured in RGM or SCGM at A1 and P1 had a substantial protein expression of these MVSC markers, then downregulated in both RGM-and SCGM-cultured cells over time. The magnitude of MVSC marker downregulation was much more dramatic in the RGM-cultured cells.(5) Freshly isolated RASMCs expressed mesenchymal stem cell markers CD29, CD90 and CD44H, and these markers did not change in RASMCs cultured in RGM by P10.2. "RASMCs" cultured in SCGM have multipotency.(1) PDGF-BB significantly induced a robust upregulation of aSMA, SM22a, calponin, and smoothelin in P2 RASMC cultured in SCGM, while RGM per se led to only a slight upregulation of aSMAand SM22a.(2) Relative to SCGM control, some genes related to matrix synthesis, such as collagen Ⅰ and Ⅲ, elastin, and matrix metallopeptidase (MMP)2/9, were upregulated in the cells cultured in RGM with PDGF.(3) The cells cultured in RGM and RGM with PDGF grew rapidly and the growth rate was positively associated with the SMC gene expression levels.(4) The SCGM-expanded P3 cells could be induced into adipocytes, chondrocytes, and osteocytes by established combinations of cytokines for adipogenesis, chondrogenic and ostogenic differentiation, respectively.3. Endogenous ROS are a negative regulator of rat aortic MVSC differentiation into synthetic VSMCs.(1) Rat aortic MVSCs had a higher level of endogenous ROS, compared with differentiated RASMCs and there was a passage-dependent decrease in the level of endogenous ROS in differentiated RASMCs.(2) In PDGF-induced VSMCs, several anti-oxidant genes including Nrf2, NQO1, GCLC, GR, and SOD2 were all upregulated. In RGM cultured MVSCs, these anti-oxidant genes were also dramatically upregulated after P3 and maintained their high expression levels up to P10.(3) In SCGM cultured P2 MVSCs, addition of ROS scavengers Tiron and NAC upregulated the cellular expression of αSMA, SM22α, calponin, and smoothelin. And the induced VSMCs exhibited an increased growth rate relative to the non-induced MVSCs cultured in SGCM.4. Phospholipase A2, group 7 (Pla2g7) is a negative regulator of rat aortic MVSC differentiation into synthetic VSMCs.(1) mRNA expression of Pla2g7 but not NEDD8 was downregulated during PDGF-induced SMC differentiation of MVSCs.(2) Knockdown of Pla2g7 but not NEDD8 enhanced MVSCs differentiate into SMCsConclusion:1. The most of the surviving RASMCs at A1-PI may be MVSCs and the expanded RASMCs in conventional RGM may be the result of a process of differentiation of MVSCs.2. Endogenous ROS is critical for the maintenance of the undifferentiated status of MVSCs and that suppression of endogenous ROS formation triggers MVSC differentiation into synthetic VSMCs.3. Pla2g7 plays an important role in maintaining an undifferentiated state of MVSCs via facilitating endogenous ROS formation.Background:Many vascular diseases are caused by endothelial cell (EC) injury and dysfunction, which occurs in chronic metabolic diseases such as metabolic syndrome and type II diabetes mellitus. In many chronic metabolic diseases, vascular endothelial integrity is affected by EC proliferation and apoptosis, which assures blood vessel function. Therefore, restoration of injured EC via regulating endothelial cell proliferation and apoptosis may have very important significance. Thus, extensive efforts were made to find more metabolic related factors that can promote endothelial cell proliferation and avoid their death, but the outcomes were not encouraging.The benefits of exercise in metabolic and cardiovascular disease prevention and progression have been well documented. Irisin is a newly discovered myokine that links exercise with increased energy expenditure to produce fundamental exercise-based health benefits. Irisin is released from skeletal muscles and is increased with exercise when the fibronectin type III domain containing 5 (Fndc5) is proteolyzed. Irisin is highly conserved across species. Irisin has been proposed to be a bridge between exercise and metabolic homeostasis and to be involved in modest weight loss and improved glucose intolerance in mice. Recent studies discovered that type 2 diabetic patients displayed significantly lower levels of circulating irisin compared with non-diabetic control subjects. Circulating irisin levels were decreased in patients with chronic kidney disease (CKD) and were independently associated with high-density lipoprotein cholesterol levels. Intriguingly, a new study demonstrated that pharmacological irisin concentrations promote mouse H19-7 HN cell proliferation via the STAT3 signaling pathway. This finding suggests that irisin may have a pro-proliferation effect in addition to its role in regulating metabolic homeostasis. However, no previous studies have evaluated whether irisin may directly regulate human EC.Objective:The aim of this study was to investigate the role of irisin on human umbilical vein endothelial cell proliferation and apoptosis, as well as possible signaling mechanisms by which irisin exerts its effects.Materials and methods:1. HUVEC culture to do the following experiments.2. The expression and purification of human recombinant irisin to do the following experiments.3. HUVEC proliferation was measured by [3H]thymidine uptake.4. HUVEC proliferation was detected by cell counting.5. Ki67 expression was detected by Immunofluorescent staining.6. Protein expression was detected by Western blot.7. HUVEC apoptosis was detected by Flow cytometry.8. Statistics:The data are expressed as the mean±standard deviation (SD). All of the experiments were repeated at least three times. Comparisons among values for all groups were performed by one-way analysis of variance (ANOVA). Holm’s t-test was used for analysis of differences between different groups. Differences were considered to be significant at P<0.05.Results:1. Effect of irisin on HUVEC proliferation.(1) The increase of [3H] thymidine uptake induced by irisin (20 nM) was 2.4 times higher than the control group in serum-free conditions.(2) The result of cell counting demonstrated that irisin (20 and 40 nM) can significantly accelerate HUVEC proliferation.(3) The Ki67 immunofluorescent staining demonstrated that there were more Ki67-expressing cells in the irisin stimulation group than the control group.2. Irisin mediates HUVEC proliferation through the ERK signaling pathway.(1) After irisin treatment of HUVECs, phosphorylated ERK (P-ERK) levels were significantly increased. And treating HUVECs with irisin had no effect on the level of phospho-p38 and AKT.(2) The r-irsin-induced increase of [3H] thymidine uptake was significantly reduced by ERK inhibitor (U0126) treatment. Similar results were observed using immunofluorescent staining.3. Irisin protects HUVECs from high glucose-induced apoptosisFlow cytometry results demonstrated that irisin effectively attenuated high glucose-induced apoptosis in HUVECs at a dose of 20 nM.4. Irisin down-regulates Bax, Caspase-9, Caspase-3 and up-regulates Bcl-2 expression in HUVECs on high glucose conditions.The Western blot results indicated that following the treatment with 20 nM irisin, expression of protein Bax, Caspase-9 and Caspase-3 decreased and the anti-apoptotic protein Bcl-2 increased. The expression of GSK-3β and Bad were not influenced.Conclusion:1. Irisin promote HUVEC proliferation.2. Irisin mediates HUVEC proliferation through the ERK signaling pathway.3. Irisin protects HUVECs from high glucose-induced apoptosis.4. Irisin down-regulates Bax, Caspase-9, Caspase-3 and up-regulates Bcl-2 expression in HUVECs on high glucose conditions.