Current Drug Targets - Volume 11, Issue 8, 2010

Worldwide research activities about the role of small regulatory RNA molecules, especially microRNAs, have been exploded and promise development of more mechanistic-orientated molecular therapies of an array of diseases. One of the most important characteristics of miRNAs may be the fact the miRNAs not only regulate single genes but fine-tune complete pathways of often co-ordinately acting genes. In this highlight issue of Current Drug Targets an in-depth overview about the current role of miRNAs in cardiovascular disease processes and the development of miRNA-based therapeutic approaches are presented and discussed. Da Costa Martins et al. review the current knowledge about the basic mechanisms by which cardiovascular miRNAs are regulated in the larger context of cardiogenesis and in cardiovascular disease [1]. Especially they discuss the role of transcription factors in regulating miRNAs and address the role of Dicer activity in the developing and mature heart. Ikeda and Pu describe and summarize changes in miRNA expression in cardiovascular diseases and specially focus on functional importance of selected miRNAs in cardiac diseases [2]. Changes in miRNA expression often lead to severe alterations of multiple cardiovascular functions both in physiological and pathophysiological settings. Latronico and Condorelli focus about recent discoveries pertaining to miRNAs as new regulators of cardiac electrophysiology and pathology [3]. The authors present an update of what is known about the regulation of ion channels and related genes by cardiovascular miRNAs and speculate on potential developments of miRNA-based therapeutic manipulation of cardiac arrhythmogenesis. As miR-21 is one of the most studied miRNAs in general, a separate chapter by Jazbutyte and Thum summarizes the current knowledge of the role of this miRNA in cardiovascular disease and in cancer, where it is also activated and serves as a potential target [4]. Next to the importance of miRNAs in heart tissue, there is also an important role in the vascular homeostasis, inflammatory processes and initiation and progression of atherosclerosis. The review by Bonauer et al. therefore summarizes the current knowledge of miRNAs in the vasculature, especially in endothelial and smooth muscle cells [5], whereas Weber and colleagues report on miRNA-mediated arterial remodelling, inflammation and atherosclerosis [6]. Finally, Montgomery and van Rooij give an overview about the current and future developments of miRNA-based therapeutic strategies in cardiovascular medicine [7]. The recent discovery of miRNAs as therapeutic targets in multiple diseases including cardiovascular diseases and cancers should be followed in the future with great passion, caution and patience. Examples have proven that miRNA manipulation can be an effective tool with potential therapeutic relevance to disease. However, intense future research is needed to answer questions about delivery, specificity, reversibility, and potential toxicity of various miRNA modulators.

The molecular biology dogma that DNA replicates its genetic information within nucleotide sequences and transcribes it to RNA where it codes for the generation of mRNA, failed to consider a significant part of the genetic code. Although it has been generally assumed that most genetic information is executed by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is transcribed into non-coding RNA (ncRNA), many of which are alternatively spliced and/or processed into smaller functional RNA molecules. ncRNAs are predominantly involved in processes that require highly specific nucleic acid recognition, revealing a, so far hidden, layer of internal signals that control various levels of gene expression in developmental and (patho)physiological processes. MicroRNAs (miRNAs) are a large class of evolutionary conserved, small ncRNAs, typically 18 to 24 nucleotides in length, that primarily function at the posttranscriptional level by interacting with the 3' untranslated region (UTR) of specific target mRNAs in a sequence-specific manner. Despite the advances in miRNA discovery, the role of miRNAs in physiological and pathological processes is just rising, revealing their cellular functions in proliferation and differentiation, apoptosis, the stress response and tumorgenesis. MiRNA expression profiling and the manipulation of their expression in cardiac tissue has led to the discovery of regulatory roles for these small ncRNAs during cardiac development and disease, implicating them in regulation of cardiac gene expression. Here we review the basic mechanisms by which cardiovascular miRNAs are regulated in the larger context of cardiogenesis and in cardiovascular disease.

Heart ion-channel function and expression are continuously being regulated on the basis of the hemodynamic state of the cardiovascular system, the neurohumoral milieu and the properties of the ongoing ionic fluxes. These homeostatic forces act through multiple mechanisms at transcriptional, translational and post-translational levels. Of clinical importance is the fact that with adverse stress these regulatory mechanisms can produce arrhythmogenic channel remodelling. Although a great deal is known about the functionality of ion channels and the generation of the action potential, much less is known about the underlying controlling mechanisms and how these become derailed during disease. microRNA-mediated posttranscriptional control is a very recent addition to cardiovascular biology. Here, we outline discoveries pertaining to these new regulators and how they might be involved in cardiac electrophysiology and pathology.

microRNAs (miRNAs) are powerful, recently recognized regulators of gene expression. miRNAs modulate virtually all aspects of cardiac biology, from cardiac specification and development to cardiomyocyte survival and hypertrophy. Expression profiling of experimental and human heart disease has shown that miRNA expression is altered in heart disease, and miRNA expression signatures may be useful biomarkers for heart disease diagnosis and prognosis. Mechanistic studies have revealed how miRNAs contribute to heart disease pathogenesis. Here we review the expression and function of miRNAs in heart disease.

MicroRNA-21 (miR-21) expression is activated in multiple types of cancers, such as breast, liver, brain, prostate, myometrial cancers but also in cardiovascular disease. MiR-21 regulates a plethora of target proteins which are involved in cellular survival, apoptosis and cell invasiveness. MiR-21 regulation is complex due to an own promoter that is target for various transcription factors and hormones. The consistent miR-21 overexpression under pathophysiological conditions points to miR-21 as a valuable tool for new therapeutic strategies. In this review, we present and analyze current data about miR-21 expression in various pathologies ranging from cancer to cardiovascular disease. Further, miR- 21 regulatory mechanisms and miR-21 downstream targets are discussed. Finally, we highlight the particular role of miR- 21 as a therapeutic target in various diseases.

MicroRNAs are small non-coding RNAs that regulate gene expression at the posttranscriptional level by either inhibiting mRNA translation or inducing mRNA degradation. These regulatory mechanisms occur in a sequence-specific manner through the direct binding of the microRNA to complementary reverse sequences in the 3' UTR of target mRNAs. The sequence-specific nature of microRNAs allows for the regulation of numerous target mRNAs, which often are related genes, resulting in the robust regulation of entire pathways. Previous studies have identified expression signatures of microRNAs during various pathological settings, including those of cardiovascular disease. As evident through gain- and loss-of-function studies in mice, it is apparent microRNAs play specific and essential roles during cardiac hypertrophy, fibrosis, angiogenesis, apoptosis, and contractility. The powerful effects of altering microRNA levels genetically have resulted in the rapid progression of oligo-based regulation of microRNAs as a new class of cardiovascular therapeutics. Here we summarize the current oligo-based technologies in use to regulate microRNA levels in vivo and how these technologies have been applied to multiple microRNAs during cardiovascular disease.

MicroRNAs are endogenously expressed small non-coding RNAs that regulate gene expression on the posttranscriptional level. During the last years microRNAs have emerged as key regulators of several physiological and pathophysiological processes in the vascular wall. Endothelial cell functions and angiogenesis are critically regulated by microRNAs such as miR-126 and the miR-17-92 cluster in vitro and in vivo. Tumor angiogenesis is additionally controlled by miR-296 and miR-378. MicroRNAs also regulate smooth muscle cell phenotypes and control neointima formation and atherosclerosis. In this respect, miR-143 and miR-145 have been shown to play a crucial role. In this review, we summarize the role of microRNAs and their target genes in endothelial and smooth muscle cells and discuss their applicability as drug targets.

Atherosclerosis is now widely appreciated to represent a chronic inflammatory reaction of the vascular wall in response to dyslipidemia and endothelial distress involving the inflammatory recruitment of leukocytes and the activation of resident vascular cells. The proliferative response of smooth muscle cells critically contributes to arterial remodelling. As part of the inflammatory infiltrate, monocytes/macrophages, but also dendritic cells, lymphocytes and neutrophils contribute to the pathogenesis of atherosclerosis. The analysis of microRNA (miR) expression in arterial lesions after balloon injury has revealed fundamental changes in the miR signature comprising many different miRs. Moreover, single miRs have been pinpointed to exert a significant impact on neointimal lesion formation. While studies addressing the profile of miR expression during the development of native atherosclerotic plaques are ongoing, it is conceivable that miRs expressed in inflammatory cell subsets would also affect disease progression. Here we summarize the role of miRs in arterial remodelling and atherosclerosis and putative roles of miRs in vascular inflammation by regulating the differentiation and functions of immune cell subsets. Given the importance of the delicately orchestrated immune response in atherosclerosis and arterial remodelling, miRs will exert profound effects during the evolution of lesion formation and constitute possible targets for therapeutic interventions.

Glucocorticoids are the mainstay of asthma management and effectively treat acute exacerbations of asthma. However, a small subset of asthmatics, usually with severe asthma, respond poorly even to systemic administration of high-dose glucocorticoids and this condition is termed “steroid-resistant asthma”. This cohort, although small, accounts for ~50% of total health care cost for asthma. New investigations into the mechanisms of glucocorticoid action have broadened and deepened our understanding of glucocorticoid resistance. Here we review the importance and characteristics of steroid resistant asthma, the mechanisms that mediate the function of glucocorticoids and that lead to the development of this disease and potential therapies to reverse resistance to treatment. Cellular and molecular factors, receptors and complex signalling pathways have all been implicated. Indeed, based on molecular biological studies, excessive activation of intracellular transcription factors, impaired histone deacetylase, and epigenetic (such as miR-18 and miR-124a) as well as other factors (e.g. vitamin D, P-glycoprotein 170, and macrophage migration inhibitory factor and T helper 17 cells and factors related to innate immunity (such as IFN-γ and LPS)) may result in glucocorticoid resistance. A thorough understanding of the pathogenesis of steroid resistant asthma will help to develop more efficacious agents for the treatment of the disease.

Kisspeptin has emerged as a critical player in the initiation of puberty and reproductive function. In humans, inactivating mutations of the kisspeptin receptor result in hypogonadotrophic hypogonadism and kisspeptin receptor activating mutations cause precocious puberty. Kisspeptin potently stimulates the release of gonadotrophins predominantly through the release of gonadotrophin-releasing hormone (GnRH). Here we review the data from animal and human studies exploring the role of kisspeptin in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. Kisspeptin signalling presents a novel target for therapeutic manipulation of the HPG axis.

The eye is innervated by numerous serotonergic nerves and serotonin (5-hydroxytryptamine; 5HT) is present in the aqueous humor of animal and human eyes. In an effort to delineate the role of the serotonergic system in modulating intraocular pressure (IOP) within the anterior segment of the eye, extensive topical ocular dosing studies were conducted with a variety of 5HT ligands and in various animal species. Even though certain 5HT1A agonists decreased IOP in rabbits, these compounds failed to affect IOP in normotensive or ocular hypertensive monkey eyes. In contrast, while 5HT2 agonists induced significant IOP reductions in normotensive rat eyes and in eyes of ocular hypertensive Cynomolgus monkeys, these agents were inactive in ocular normotensive cats and rabbits. Additional studies indicated a strong involvement of 5HT2A receptors in mediating IOP-lowering in conscious ocular hypertensive Cynomolgus monkeys. As a result of further structure-activity investigations, AL-34662, a selective 5HT2 agonist (relative to other 5HT receptor types and sub-types) with high affinity, potency and efficacy at 5HT2A, 5HT2B and 5HT2C receptors was discovered that efficaciously lowered IOP in the monkey model of ocular hypertension (33 ± 3 % reduction out to 6 hrs post with a 300 μg topical ocular dose). Due to unavailability of monkey ocular cells, extensive in vitro studies were conducted using relevant human ocular cells in order to correlate with and support the in vivo observations in the monkeys. RT-PCR and in situ hybridization studies revealed the presence of mRNAs for 5HT2A-C receptor subtypes in human ocular tissues involved in IOP modulation. The relative distribution and density of these mRNAs were as follows: ciliary body (CB) (5HT2A > 5HT2B > 5HT2C), ciliary epithelium (CE) (5HT2A > 5HT2B = 5HT2C) and trabecular meshwork (TM) (5HT2A= 5HT2B >> 5HT2C). Furthermore, quantitative autoradiography revealed a relatively high specific binding of [3H]-5HT and [3H]-ketanserin to 5HT2 receptors in human CE and longitudinal ciliary muscle (CM). Second messenger studies revealed the presence of phospholipase C-coupled 5HT2A receptors in h-CM and h-TM cells where they stimulated phosphoinositide (PI) hydrolysis and mobilized intracellular Ca2+ when challenged with a variety of 5HT2A-C receptor agonists (e.g. α-methyl-5HT, (R)-DOI, α-methyl-5HT, BW-723C86, MK-212, mCPP, cabergoline, AL-34662). These functional responses were blocked by selective 5HT2 receptor antagonists with the 5HT2A antagonist, M-100970, exhibiting the highest potency. Thus, functional 5HT2A receptors are present in human ocular cells involved in IOP reduction and this correlates with the ability of 5HT2A agonists to lower IOP in Cynomolgus monkeys, a surrogate for human subjects.

Here we review phospho-specific, quantitative flow cytometry approach as a rapid and reliable tool for measuring intracellular signaling proteins with potential applications in monitoring efficacy of targeted therapy. The single cell, multiparameter nature of flow cytometry allows simultaneous investigation of specific cell type and the corresponding intracellular markers. Peripheral blood can be directly stained with surface markers to delineate cell populations of interest, followed by fixation, permeabilization, and immunostaining with specific antibodies to the cellular targets. By using calibrated standardized phycoerythrin (PE)-conjugated beads for signal quantification, an informative Index value can be generated for each sample by multiplication of percentages of positive cells with fluorescence intensity per cell. This technique can yield both qualitative and quantitative information on effects of cellular markers upon targeted therapy, thereby providing another layer of advantages over the conventional flow cytometry analysis. Advances in this technology: high-throughput capability and automation, making it a valuable platform in modern drug discovery.

New blood vessel formation (angiogenesis) is fundamental to tumor growth, invasion, and metastatic dissemination. The vascular endothelial growth factor (VEGF) signaling pathway plays pivotal roles in regulating tumor angiogenesis. VEGF as a therapeutic target has been validated in various types of human cancers. Different agents including antibodies, aptamers, peptides, and small molecules have been extensively investigated to block VEGF and its pro-angiogenic functions. Some of these agents have been approved by FDA and some are currently in clinical trials. Combination therapies are also being pursued for better tumor control. By providing comprehensive real-time information, molecular imaging of VEGF pathway may accelerate the drug development process. Moreover, the imaging will be of great help for patient stratification and therapeutic effect monitoring, which will promote effective personalized molecular cancer therapy. This review summarizes the current status of tumor therapeutic agents targeting to VEGF and the applications of VEGF related molecular imaging.

Aberrant folded proteins are hallmarks of amyloidogenic diseases. Examples are Alzheimer's disease (AD) and prion-related disorders (PrD). These disorders, although clinically different, have the same underlying pathogenetic mechanism: an altered protein conformer with high β-sheet structure content: the amyloid beta peptide (Aβ) in the case of AD, and the aberrant prion protein, PrPsc in PrD. Although the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, there is good reason to expect prion research to profit from advances in the understanding of AD, and vice versa. Growing evidence indicates that the various pathways of lipid/lipoprotein metabolism play a key role in AD and PrD pathophysiology. These findings clearly highlight the possible involvement of cholesterol in misfolded protein generation. In this review, we focus on recent studies which provide evidence that membrane domains, called lipid rafts, directly promote protein misfolding, and that this process takes place only if changes occur in the fine regulation of intracellular cholesterol. In addition, we discuss the implications of these results to introduce the concept that pharmacological interventions restoring cholesterol homeostasis could have potential preventive/therapeutic value against the progression of misfolding disorders. The aim of the review is to provide researchers with a general understanding of cholesterol's involvement in protein folding/misfolding processes which may be relevant for knowledge advancement regarding amyloidogenic proteins, and possible ways to prevent their pathological activity.

Heart failure, the common end-point of many cardiac diseases, is a major contributor to mortality and morbidity and contributes considerably to health care costs. Current treatment regimens include β-adrenergic antagonists, angiotensin converting enzyme inhibitors, and inotropic agents are used by some patients. Studies in experimental animals have demonstrated that inhibition of signaling pathways downstream of the heterotrimeric G protein Gq reduces ventricular hypertrophy and protects from the development of heart failure. However, targets identified, to date, have been limited by a lack of tissue specificity. In cardiomyocytes, Gq activates only one splice variant of one subtype of phospholipase Cβ, specifically phospholipase Cβ1b (PLCβ1b) and PLCβ1b is responsible for Gq mediated hypertrophic and apoptotic responses. PLCβ1b targets the sarcolemma via its unique C-terminal sequence and its activation can be inhibited by expressing the C-terminal sequence to compete for sarcolemmal binding. Inhibition of PLCβ1b by the C-terminal peptide reduces hypertrophic responses in cardiomyocytes. We present the evidence that inhibition of the sarcolemmal association of PLCβ1b provides a cardiac-specific target for the development of drugs to reduce pathological cardiac hypertrophy and thereby to reduce the burden of heart failure.