Flank sequences of miR-145/143 and their aberrant expression in vascular disease: mechanism and therapeutic application.

BACKGROUND: Many microRNAs (miRNAs) are downregulated in proliferative vascular disease. Thus, upregulation of these miRNAs has become a major focus of research activity. However, there is a critical barrier in gene therapy to upregulate some miRNAs such as miR-145 and miR-143 because of their significant downregulation by the unclear endogenous mechanisms under disease conditions. The purpose of this study was to determine the molecular mechanisms responsible for their downregulation and to overcome the therapeutic barrier. METHODS AND RESULTS: In cultured proliferative rat vascular smooth muscle cells (VSMCs) in vitro and in diseased rat and mouse arteries in vivo, we have identified that the impairment of pri-miR-145 into pre-miR-145 is the critical step related to the downregulation of miR-145, in which the PI3-kinase/Akt/p53 pathway is involved. We further identified that the flank sequences of pri-miR-145 are the critical structural components responsible for the aberrant miR-145 expression. Switching of the flank sequence of downregulated miR-145 and miR-143 to the flank sequence of miR-31 confers resistance to their downregulation. The genetically engineered miR-145 (smart miR-145) restored the downregulated miR-145 in proliferative rat VSMCs and in rat carotid arteries with balloon injury and mouse atherosclerotic aortas and demonstrated much better therapeutic effects on the abnormal growth of VSMCs, expression of its target gene, KLF5 expression, VSMC marker gene expression, and vascular neointimal growth. CONCLUSIONS: The flank sequences of miR-145 and miR-143 play a critical role in their aberrant expression in VSMCs and vascular walls. The genetically engineered "smart" miRNAs based on their flank sequences may have broadly therapeutic applications for many vascular diseases.

An essential role of PDCD4 in vascular smooth muscle cell apoptosis and proliferation: implications for vascular disease.

It is well established that vascular smooth muscle cell (VSMC) apoptosis and proliferation are critical cellular events in a variety of human vascular diseases. However, the molecular mechanisms involved in controlling VSMC apoptosis and proliferation are still unclear. In the current study, we have found that programmed cell death 4 (PDCD4) is significantly downregulated in balloon-injured rat carotid arteries in vivo and in platelet-derived growth factor-stimulated VSMCs in vitro. Overexpression of PDCD4 via adenovirus (Ad-PDCD4) increases VSMC apoptosis in an apoptotic model induced by serum deprivation. In contrast, VSMC apoptosis is significantly decreased by knockdown of PDCD4 via its small interfering RNA. In the rat carotid arteries in vivo, VSMC apoptosis is increased by Ad-PDCD4. We have further identified that activator protein 1 is a downstream signaling molecule of PDCD4 that is associated with PDCD4-mediated effects on VSMC apoptosis. In addition, VSMC proliferation was inhibited by overexpression of PDCD4. The current study has identified, for the first time, that PDCD4 is an essential regulator of VSMC apoptosis and proliferation. The downregulation of PDCD4 expression in diseased vascular walls may be responsible for the imbalance of VSMC proliferation and apoptosis. The results indicate that PDCD4 may be a new therapeutic target 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.

Vasoactive intestinal polypeptide entrains circadian rhythms in astrocytes.

Many mammalian cell types show daily rhythms in gene expression driven by a circadian pacemaker. For example, cultured astrocytes display circadian rhythms in Period1 and Period2 expression. It is not known, however, how or which intercellular factors synchronize and sustain rhythmicity in astrocytes. Because astrocytes are highly sensitive to vasoactive intestinal polypeptide (VIP), a neuropeptide released by neurons and important for the coordination of daily cycling, the authors hypothesized that VIP entrains circadian rhythms in astrocytes. They used astrocyte cultures derived from knock-in mice containing a bioluminescent reporter of PERIOD2 (PER2) protein, to assess the effects of VIP on the rhythmic properties of astrocytes. VIP induced a dose-dependent increase in the peak-to-trough amplitude of the ensemble rhythms of PER2 expression with maximal effects near 100 nM VIP and threshold values between 0.1 and 1 nM. VIP also induced dose- and phase-dependent shifts in PER2 rhythms and daily VIP administration entrained bioluminescence rhythms of astrocytes to a predicted phase angle. This is the first demonstration that a neuropeptide can entrain glial cells to a phase predicted by a phase-response curve. The authors conclude that VIP potently entrains astrocytes in vitro and is a candidate for coordinating daily rhythms among glia in the brain.