Yunhui Cheng

MicroRNA Expression Signature and Antisense-Mediated Depletion Reveal an Essential Role of MicroRNA in Vascular Neointimal Lesion Formation

MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21–mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21–mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.

MicroRNA-145, a Novel Smooth Muscle Cell Phenotypic Marker and Modulator, Controls Vascular Neointimal Lesion Formation

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. Recently, we have found that microRNA (miRNA) miR-145 is the most abundant miRNA in normal vascular walls and in freshly isolated VSMCs; however, the role of miR-145 in VSMC phenotypic modulation and vascular diseases is currently unknown. Here we find that miR-145 is selectively expressed in VSMCs of the vascular wall and its expression is significantly downregulated in the vascular walls with neointimal lesion formation and in cultured dedifferentiated VSMCs. More importantly, both in cultured rat VSMCs in vitro and in balloon-injured rat carotid arteries in vivo, we demonstrate that the noncoding RNA miR-145 is a novel phenotypic marker and a novel phenotypic modulator of VSMCs. VSMC differentiation marker genes such as SM α-actin, calponin, and SM-MHC are upregulated by premiR-145 or adenovirus expressing miR-145 (Ad-miR-145) but are downregulated by the miR-145 inhibitor 2′OMe-miR-145. We have further identified that miR-145–mediated phenotypic modulation of VSMCs is through its target gene KLF5 and its downstream signaling molecule, myocardin. Finally, restoration of miR-145 in balloon-injured arteries via Ad-miR-145 inhibits neointimal growth. We conclude that miR-145 is a novel VSMC phenotypic marker and modulator that is able of controlling vascular neointimal lesion formation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.

A Necessary Role of miR-221 and miR-222 in Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia

MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression. Functionally, an individual miRNA is as important as a transcription factor because it is able to regulate the expression of its multiple target genes. Recently, miR-221 and miR-222 have been found to play a critical role in cancer cell proliferation. However, their roles in vascular smooth muscle cell (VSMC) biology are currently unknown. In the present study, the time course changes and cellular distribution of miR-221 and miR-222 expression were identified in rat carotid arteries after angioplasty, in which their expression was upregulated and localized in VSMCs in the injured vascular walls. In cultured VSMCs, miR-221 and miR-222 expression was increased by growth stimulators. Knockdown of miR-221 and miR-222 resulted in decreased VSMC proliferation in vitro. Using both gain-of-function and loss-of-function approaches, we found that p27(Kip1) and p57(Kip2) were 2 target genes that were involved in miR-221- and miR-222-mediated effect on VSMC growth. Finally, knockdown of miR-221 and miR-222 in rat carotid arteries suppressed VSMC proliferation in vivo and neointimal lesion formation after angioplasty. The results indicate that miR-221 and miR-222 are novel regulators for VSMC proliferation and neointimal hyperplasia. These findings may also represent promising therapeutic targets in proliferative vascular diseases.

MicroRNA Expression Signature and the Role of MicroRNA-21 in the Early Phase of Acute Myocardial Infarction

Several recent reports have suggested that microRNAs (miRNAs) might play critical roles in acute myocardial infarction (AMI). However, the miRNA expression signature in the early phase of AMI has not been identified. In this study, the miRNA expression signature was investigated in rat hearts 6 h after AMI. Compared with the expression signature in the noninfarcted areas, 38 miRNAs were differentially expressed in infarcted areas and 33 miRNAs were aberrantly expressed in the border areas. Remarkably, miR-21 expression was significantly down-regulated in infarcted areas, but was up-regulated in border areas. The down-regulation of miR-21 in the infarcted areas was inhibited by ischemic preconditioning, a known cardiac protective method. Overexpression of miR-21 via adenovirus expressing miR-21 (Ad-miR-21) decreased myocardial infarct size by 29% at 24 h and decreased the dimension of left ventricles at 2 weeks after AMI. Using both gain-of-function and loss-of-function approaches in cultured cardiac myocytes, we identified that miR-21 had a protective effect on ischemia-induced cell apoptosis that was associated with its target gene programmed cell death 4 and activator protein 1 pathway. The protective effect of miR-21 against ischemia-induced cardiac myocyte damage was further confirmed in vivo by decreased cell apoptosis in the border and infarcted areas of the infarcted rat hearts after treatment with Ad-miR-21. The results suggest that miRNAs such as miR-21 may play critical roles in the early phase of AMI.

MicroRNA-21 protects against the H2O2-induced injury on cardiac myocytes via its target gene PDCD4

Reactive oxygen species (ROS)-induced cardiac cell injury via expression changes of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate about 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. Currently, the effects of ROS on miRNA expression and the roles of miRNAs in ROS-mediated injury on cardiac myocytes are uncertain. Using quantitative real-time RT-PCR (qRT-PCR), we demonstrated that microRNA-21 (miR-21) was upregulated in cardiac myocytes after treatment with hydrogen peroxide (H(2)O(2)). To determine the potential roles of miRNAs in H(2)O(2)-mediated gene regulation and cellular injury, miR-21 expression was downregulated by miR-21 inhibitor and upregulated by pre-miR-21. H(2)O(2)-induced cardiac cell death and apoptosis were increased by miR-21 inhibitor and was decreased by pre-miR-21. Programmed cell death 4 (PDCD4) that was regulated by miR-21 and was a direct target of miR-21 in cardiac myocytes. Pre-miR-21-mediated protective effect on cardiac myocyte injury was inhibited in H(2)O(2)-treated cardiac cells via adenovirus-mediated overexpression of PDCD4 without miR-21 binding site. Moreover, Activator protein 1 (AP-1) was a downstream signaling molecule of PDCD4 that was involved in miR-21-mediated effect on cardiac myocytes. The results suggest that miR-21 is sensitive to H(2)O(2) stimulation. miR-21 participates in H(2)O(2)-mediated gene regulation and functional modulation in cardiac myocytes. miR-21 might play an essential role in heart diseases related to ROS such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial ischemia/reperfusion injury.

A translational study of circulating cell-free microRNA-1 in acute myocardial infarction.

miRNAs (microRNAs) participate in many diseases including cardiovascular disease. In contrast with our original hypothesis, miRNAs exist in circulating blood and are relatively stable due to binding with other materials. The aim of the present translational study is to establish a method of determining the absolute amount of an miRNA in blood and to determine the potential applications of circulating cell-free miR-1 (microRNA-1) in AMI (acute myocardial infarction). The results revealed that miR-1 is the most abundant miRNA in the heart and is also a heart- and muscle-specific miRNA. In a cardiac cell necrosis model induced by Triton X-100 in vitro, we found that cardiac miR-1 can be released into the culture medium and is stable at least for 24 h. In a rat model of AMI induced by coronary ligation, we found that serum miR-1 is quickly increased after AMI with a peak at 6 h, in which an increase in miR-1 of over 200-fold was demonstrated. The miR-1 level returned to basal levels at 3 days after AMI. Moreover, the serum miR-1 level in rats with AMI had a strong positive correlation with myocardial infarct size. To verify further the relationship between myocardial size and miR-1 level, an IP (ischaemic preconditioning) model was used. The results showed that IP significantly reduced circulating miR-1 levels and myocardial infract size induced by I/R (ischaemia/reperfusion) injury. Finally, the levels of circulating cell-free miR-1 were significantly increased in patients with AMI and had a positive correlation with serum CK-MB (creatine kinase-MB) levels. In conclusion, the results suggest that serum miR-1 could be a novel sensitive diagnostic biomarker for AMI.

Ischaemic preconditioning-regulated miR-21 protects heart against ischaemia/reperfusion injury via anti-apoptosis through its target PDCD4

AIMS The aims of the present study are to determine the miRNA expression signature in rat hearts after ischaemic preconditioning (IP) and to identify an IP-regulated miRNA, miR-21, in IP-mediated cardiac protection, and the potential cellular and molecular mechanisms involved. METHODS AND RESULTS The miRNA expression signature was investigated in rat hearts. Among the 341 arrayed miRNAs, 40 miRNAs were differentially expressed (21 up and 19 down) in rat hearts with IP, compared with their controls. Some of these differentially expressed miRNAs were further verified by quantitative reverse transcriptase-polymerase chain reaction. Remarkably, miR-21 was one of most upregulated miRNAs in hearts after IP. In vivo, IP-mediated cardiac protection against ischaemia/reperfusion injury was inhibited by knockdown of cardiac miR-21. In cultured cardiac myocytes, we identified that miR-21 also had a protective effect on hypoxia/reoxygenation-induced cell apoptosis that was associated with its target gene, programmed cell death 4. The protective effect of miR-21 on cardiac cell apoptosis was further confirmed in rat hearts after ischaemia/reperfusion injury in vivo. CONCLUSION The results suggest that miRNAs are involved in IP-mediated cardiac protection. Identifying the roles of IP-regulated miRNAs in cardiac protection may provide novel therapeutic and preventive targets for ischaemic heart disease.

MicroRNA-21 in Cardiovascular Disease

MicroRNA-21 (miR-21) is a highly expressed microRNA (miRNA) in cardiovascular system. Recent studies have revealed that its expression is deregulated in heart and vasculature under cardiovascular disease conditions such as proliferative vascular disease, cardiac hypertrophy and heart failure, and ischemic heart disease. miR-21 is found to play important roles in vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions. Accordingly, miR-21 is proven to be involved in the pathogenesis of the above-mentioned cardiovascular diseases as demonstrated by both loss-of-function and gain-of-function approaches. Programmed cell death 4 (PDCD4), phosphatase and tensin homology deleted from chromosome 10 (PTEN), sprouty1 (SPRY1), and sprouty2 (SPRY2) are the current identified target genes of miR-21 that are involved in miR-21-mediated cardiovascular effects. miR-21 might be a novel therapeutic target in cardiovascular diseases. This review article summarizes the research progress regarding the roles of miR-21 in cardiovascular disease.

Involvement of MicroRNAs in Hydrogen Peroxide-mediated Gene Regulation and Cellular Injury Response in Vascular Smooth Muscle Cells

MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate ∼30% of genes in a cell via degradation or translational inhibition of their target mRNAs. However, the effects of reactive oxygen species (ROS) on miRNA expression and the roles of miRNAs in ROS-mediated gene regulation and biological functions of vascular cells are unclear. Using microarray analysis, we demonstrated that miRNAs are aberrantly expressed in vascular smooth muscle cells (VSMCs) after treatment with hydrogen peroxide (H2O2). H2O2-mediated up-regulation of microRNA-21 (miR-21) was further confirmed by quantitative real-time PCR. To determine the potential roles of miRNAs in H2O2-mediated gene regulation and cellular effects, miR-21 expression was down-regulated by miR-21 inhibitor and up-regulated by pre-miR-21. H2O2-induced VSMC apoptosis and death were increased by miR-21 inhibitor and decreased by pre-miR-21. Programmed cell death 4(PDCD4) was a direct target of miR-21 that was involved in miR-21-mediated effects on VSMCs. Pre-miR-21-mediated protective effect on VSMC apoptosis and death was blocked via adenovirus-mediated overexpression of PDCD4 without the miR-21 binding site. Moreover, activator protein 1 was a downstream signaling molecule of PDCD4 in miR-21-modulated VSMCs. The results suggest that miRNAs in VSMCs are sensitive to H2O2 stimulation. miRN-21 participates in H2O2-mediated gene regulation and cellular injury response through PDCD4 and the activator protein 1 pathway. miRNAs might play a role in vascular diseases related to ROS.

Cell-specific effects of miR-221/222 in vessels: Molecular mechanism and therapeutic application

MicroRNAs (miRNAs) are noncoding RNAs that impact almost every aspect of biology and disease. Until now, the cell-specific effects of miRNAs in cardiovascular system have not been established. In the current study, the cellular functions of miR-221 and miR-222 (miR-221/222) in vascular smooth muscle cells (VSMCs) and vascular endothelial cells (ECs) were compared. In cultured cells, we identified that the effects of miR-221/222 on proliferation, migration, and apoptosis are opposite between VSMCs and ECs. In VSMCs, miR-221/222 had effects of pro-proliferation, pro-migration, and anti-apoptosis. In contrast, miR-221/222 had effects of anti-proliferation, anti-migration, and pro-apoptosis in ECs. The different expression profiles of their target genes, p27(Kip1), p57(kip2), and c-kit between the two cell types might be related to the opposite effects. Finally, the opposite cellular effects of miR-221/222 were verified in vivo in balloon-injured rat carotid artery as demonstrated by different consequences in neointimal growth and re-endothelialization. The results suggest that the biological functions of miR-221/222 in vascular walls are cell-specific. The opposite cellular effects of miR-221/222 on VSMCs and ECs may have important therapeutic applications in many vascular diseases such as atherosclerosis and restenosis after angioplasty.