[Show abstract][Hide abstract] ABSTRACT: Outcome varies among patients with subarachnoid hemorrhage but known prognostic factors explain only a small portion of the variation in outcome. We hypothesized that individual genetic variations influence brain and vascular responses to subarachnoid hemorrhage and investigated this using inbred strains of mice.
Preview · Article · Oct 2015 · Journal of Cerebral Blood Flow & Metabolism
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular diseases are commonly thought to be complex, non-Mendelian diseases that are influenced by genetic and environmental factors. A growing body of evidence suggests that epigenetic pathways play a key role in vascular biology and might be involved in defining and transducing cardiovascular disease inheritability. In this review, we argue the importance of epigenetics in vascular biology, especially from the perspective of endothelial cell phenotype. We highlight and discuss the role of epigenetic modifications across the transcriptional unit of protein-coding genes, especially the role of intragenic chromatin modifications, which are underappreciated and not well characterized in the current era of genome-wide studies. Importantly, we describe the practical application of epigenetics in cardiovascular disease therapeutics.
No preview · Article · Sep 2015 · Arteriosclerosis Thrombosis and Vascular Biology
[Show abstract][Hide abstract] ABSTRACT: Remarkable new advances have been made in the field of posttranscriptional gene regulation over recent years. These include the revelation of noncoding RNAs, such as microRNAs, antisense transcripts and their interactions with RNA-binding proteins (RBPs) in the context of both health and disease settings, such as hypoxia. In particular, these discoveries bear much relevance to the field of vascular biology, which historically has focused upon transcriptional processes. Thus, the contributions of these posttranscriptional gene regulatory mechanisms to vascular and endothelial biology represent a newer concept that warrants discussion.
Recent studies have revealed two emerging themes that are critical to endothelial/vascular biology and function. First is the functional integration between the microRNA pathway and the cellular hypoxic response, which, in addition to specific microRNAs, involves key components of the microRNA biogenesis machinery. A key concept here is the regulation of a master transcriptional programme through posttranscriptional mechanisms. The second major theme involves the dynamic interactions between RBPs, microRNAs and antisense RNAs. The condition-dependent collaborations and competitions between these different classes of posttranscriptional regulators reveal a critical layer of control for gene expression.
Taken together, these findings bear significant diagnostic and therapeutic implications for vascular disease.
No preview · Article · Mar 2015 · Current Opinion in Hematology
[Show abstract][Hide abstract] ABSTRACT: Binding of the receptor CXCR4 to its ligand SDF-1 promotes cell survival and is under the influence of a number of regulatory processes including enzymatic ligand inactivation by endopeptidases such as matrix metalloproteinase-9 (MMP-9). In light of the pivotal role that the SDF-1/CXCR4 axis plays in renal development and in the pathological growth of renal cells, we explored the function of this pathway in diabetic rats and in biopsies from patients with diabetic nephropathy, hypothesizing that the pro-survival effects of CXCR4 in resident cells would attenuate renal injury. Renal CXCR4 expression was observed to be increased in diabetic rats, whereas antagonism of the receptor unmasked albuminuria and accelerated tubular epithelial cell death. In cultured cells, CXCR4 blockade promoted tubular cell apoptosis, upregulated Bcl-2-associated death promoter and prevented high glucose/SDF-1 augmented phosphorylation of the pro-survival kinase, Akt. Although CXCR4 expression was also increased in biopsy tissue from patients with diabetic nephropathy serine 339 phosphorylation of the receptor, indicative of ligand-engagement, was unaffected. Coincident with these changes in receptor expression but not activity, MMP-9 was also upregulated in diabetic nephropathy biopsies. Supporting a ligand-inactivating effect of the endopeptidase, exposure of cultured cells to recombinant MMP-9 abrogated SDF-1 induced Akt phosphorylation. These observations demonstrate a potentially reno-protective role for CXCR4 in diabetes that is impeded in its actions in the human kidney by the coincident upregulation of ligand-inactivating endopeptidases. Therapeutically intervening in this interplay may limit tubulointerstitial injury, the principal determinant of renal decline in diabetes.
[Show abstract][Hide abstract] ABSTRACT: The discovery of RNA interference (RNAi) holds the potential to alter the paradigm of medical therapeutics. With the ability to selectively silence the function of a gene, RNAi not only provides an indispensable research tool for determining the function of a gene, but also offers potential for the development of novel therapeutics that will inhibit specific genes involved in disease. New concepts in therapeutics have been uncovered through the study of RNAi. Nuances have emerged. For instance, global RNAi pathways can be affected by somatic mutations in cancer and cellular stress, such as hypoxia. Also, viral gene therapy can have unexpected effects on endogenous short noncoding RNA pathways. Therefore, it is important to understand where RNAi therapeutics enter the processing pathways. We highlight the evolving use of RNAi as a new class of therapeutics, such as for amyloidosis, and address some of the anticipated challenges associated with its clinical application. This article is protected by copyright. All rights reserved.
No preview · Article · Nov 2014 · Clinical Pharmacology & Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) production is catalyzed by three distinct enzymes, namely, neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). The production of NO by vascular endothelium relies mainly on eNOS. Curiously, iNOS and nNOS also are relevant for vascular NO production in certain settings. By relaxing vascular smooth muscle, the classical view is that NO participates in O2 homeostasis by increasing local blood flow and O2 delivery. It is now appreciated that NO has an even more fundamental role in cellular oxygen sensing at the cellular and physiological level. A key component of cellular oxygen sensing is the hypoxia-inducible factor (HIF) that activates a transcriptional program to promote cellular survival under conditions of inadequate oxygen supply. Important new insights demonstrate that HIF protein is stabilized by two parallel pathways: (1) a decrease in the O2-dependent prolyl hydroxylation of HIF and (2) NO-dependent S-nitrosylation of HIF pathway components including HIF-α. The need for these two complementary pathways to HIF activation arises because decreased oxygen delivery can occur not only by decreased ambient oxygen but also by decreased blood oxygen-carrying capacity, as with anemia. In turn, NO production is tightly linked to O2 homeostasis. O2 is a key substrate for the generation of NO and impacts the enzymatic activity and expression of the enzymes that catalyze the production of NO, the nitric oxide synthases. These relationships manifest in a variety of clinical settings ranging from the unique situation of humans living in hypoxic environments at high altitudes to the common scenario of anemia and the use of therapeutics that can bind or release NO.
[Show abstract][Hide abstract] ABSTRACT: Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ specific Hb thresholds for tissue hypoxia, HIF-α (ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50g/L, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and -2α increased earlier (Hb~70-90g/L) in response to anemia. Liver also exhibited an early HIF-1α response. Carotid blood flow was increased early (Hb~70g/L) but renal blood flow remained relatively constant, only increased at Hb of 50g/L. Anemia increased nNOS (brain and kidney) and eNOS (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS-independent. HIF-dependent RNA levels increased linearly (~10 fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (~100 fold). Plasma EPO levels increased near Hb threshold of 90g/L, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) have become a powerful tool in the identification of disease-associated variants. Unfortunately, many of these studies have found that the estimated variability in cardiovascular disease risk cannot be fully explained by traditional paradigms of genetic variation in protein coding genes. Moreover, traditional views do not sufficiently explain the well-known link between cardiovascular disease and environmental influence. We posit that epigenetics, defined as chromatin-based mechanisms important in the regulation of gene expression that do not involve changes in the DNA sequence per se, represents the missing link. The nuclear-based mechanisms that contribute to epigenetic gene regulation can be broadly separated into three unique but highly interrelated processes: DNA methylation and hydroxymethylation; histone density and post-translational modifications; and RNA-based mechanisms. Together they complement the cis/trans perspective on transcriptional control paradigms in blood vessels. Moreover, it provides a molecular basis for understanding how the environment impacts the genome to modify cardiovascular disease risk over the lifetime of a cell and its offspring. This review provides an introduction to epigenetic function and cardiovascular disease, with a focus on endothelial cell biology. Additionally, we highlight emerging concepts on epigenetic gene regulation that are highly relevant to atherosclerosis and coronary artery disease.
[Show abstract][Hide abstract] ABSTRACT: The progressive decline of renal function in chronic kidney disease (CKD) is characterized by both disruption of the microvascular architecture and the accumulation of fibrotic matrix. One angiogenic pathway recently identified as playing an essential role in renal vascular development is the stromal cell-derived factor-1α (SDF-1)/CXCR4 pathway. Because similar developmental processes may be recapitulated in the disease setting, we hypothesized that the SDF-1/CXCR4 system would regulate microvascular health in CKD. Expression of CXCR4 was observed to be increased in the kidneys of subtotally nephrectomized (SNx) rats and in biopsies from patients with secondary focal segmental glomerulosclerosis (FSGS), a rodent model and human correlate both characterized by aberration of the renal microvessels. A reno-protective role for local SDF-1/CXCR4 signaling was indicated by i) CXCR4-dependent glomerular eNOS activation following acute SDF-1 administration; and ii) acceleration of renal function decline, capillary loss and fibrosis in SNx rats treated with chronic CXCR4 blockade. In contrast to the upregulation of CXCR4, SDF-1 transcript levels were decreased in SNx rat kidneys as well as in renal fibroblasts exposed to the pro-fibrotic cytokine transforming growth factor β (TGF-β), the latter effect being attenuated by histone deacetylase inhibition. Increased renal SDF-1 expression was, however, observed following the treatment of SNx rats with the ACE inhibitor, perindopril. Collectively, these observations indicate that local SDF-1/CXCR4 signaling functions to preserve microvascular integrity and prevent renal fibrosis. Augmentation of this pathway, either purposefully or serendipitously with either novel or existing therapies, may attenuate renal decline in CKD.
[Show abstract][Hide abstract] ABSTRACT: Posttranscriptional regulation of mRNA species represents a major regulatory checkpoint in the control of gene expression. Historically, RNA-binding proteins (RBPs) have been regarded as the primary regulators of mRNA stability and translation. More recently, however, microRNAs have emerged as a class of potent and pervasive posttranscriptional rheostats that similarly affect mRNA stability and translation. The observation that both microRNAs and RBPs regulate mRNA stability and translation has initiated a newer area of research that involves the examination of dynamic interactions between these two important classes of posttranscriptional regulators, the myriad of factors that influence these biological interactions, and ultimately, their effects on target mRNAs. Specifically, microRNAs and RBPs can act synergistically to effect mRNA destabilization and translational inhibition. They can also engage in competition with each other and exert opposing effects on target mRNAs. To date, several key studies have provided critical details regarding the mechanisms and principles of interaction between these molecules. Additionally, these findings raise important questions regarding the regulation of these interactions, including the roles of posttranslational modification, subcellular localization, target inhibition versus activation, and changes in expression levels of these regulatory factors, especially under stimulus- and cell-specific conditions. Indeed, further experimentation is warranted to address these key issues that pertain to the collaboration and competition between microRNAs and RBPs. Significantly, the elucidation of these important details bears critical implications for disease management, especially for those diseases in which these cellular factors are dysregulated.
[Show abstract][Hide abstract] ABSTRACT: Adult bone marrow-derived cells can improve organ function in chronic disease models, ostensibly by the release of paracrine factors. It has, however, been difficult to reconcile this prevailing paradigm with the lack of cell retention within injured organs and their rapid migration to the reticulo-endothelial system. Here we provide evidence that the salutary anti-fibrotic effects of bone marrow-derived early outgrowth cells (EOCs) are more consistent with an endocrine mode of action, demonstrating not only the presence of anti-fibrotic factors in the plasma of EOC-treated rats, but also that EOC conditioned medium (EOC-CM) potently attenuates both TGF-β and angiotensin II-induced fibroblast collagen production in vitro. To examine the therapeutic relevance of these findings in vivo, 5/6 subtotally nephrectomized (SNX) rats, a model of chronic kidney and heart failure characterized by progressive fibrosis of both organs, were randomized to receive intravenous injections of EOC-CM, unconditioned medium or 10(6) EOCs. Rats that received unconditioned medium developed severe kidney injury with cardiac diastolic dysfunction. In comparison, EOC-CM-treated rats demonstrated substantially improved renal and cardiac function and structure, mimicking the changes found in EOC-treated animals. Mass spectrometric analysis of EOC-CM identified proteins that regulate cellular functions implicated in fibrosis. These results indicate that EOCs secrete soluble factor(s) with highly potent anti-fibrotic activity, that when injected intravenously replicate the salutary effects of the cells themselves. Together, these findings suggest that an endocrine mode of action may underlie the effectiveness of cell therapy in certain settings and portend the possibility for systemic delivery of cell-free therapy. Stem Cells 2013.
[Show abstract][Hide abstract] ABSTRACT: Shiga toxin-producing E. coli represents a significant global health concern, especially as hypervirulent pathogens surface amidst outbreaks of hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is key in the microangiopathic events underlying the disease and its central role is underscored by the unprecedented HUS outbreak in Germany in 2011. The mechanisms of Stx-mediated endothelial dysfunction have been a major focus of research that has contributed to the current understanding of the pathogenic changes in endothelial phenotype leading to HUS. Among the newer concepts are Stx-mediated gene regulation in the absence of protein synthesis inhibition, a potential role for complement activation, and accumulating evidence for detectable serum markers before the onset of the classic clinical features of HUS. Further investigation of newer therapeutic targets and potential prognostic markers is essential to assess their utility in mitigating disease and/or predicting outcomes and will provide an improved overall understanding of HUS pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli O157:H7-associated hemolytic-uremic syndrome (HUS) is characterized by profound prothrombotic abnormalities. Endothelial
dysfunction, manifested as dysregulation of angiopoietins 1 and 2 (Ang-1/2), could underlie HUS pathophysiology. We measured
Ang-1/2 in 77 children with E. coli O157:H7 infection. Ang-1, Ang-2, and the Ang-2/Ang-1 ratio were significantly different in HUS vs the pre-HUS phase of illness
or uncomplicated infection. Angiopoietin dysregulation preceded HUS and worsened as HUS developed. In vitro exposure of human
microvascular endothelial cells to Shiga toxin recapitulated the in vivo observations. Angiopoietin regulation is profoundly
affected before and during HUS, reflecting that subclinical endothelial dysfunction precedes overt microangiopathy.
Full-text · Article · Jun 2013 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: The human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Post-transcriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially in hypoxic conditions. Here we show that the human eNOS 3' -untranslated region (3' -UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA-EMSAs revealed the formation of hnRNP E1-containing ribonucleoprotein (RNP) complexes at these 3' -UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE, 3' -UTR-targeting siRNAs, as well as microRNAs, specifically hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3' -UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the post-transcriptional inhibitory effects of antisense RNA and microRNAs under basal conditions, but is disrupted under hypoxic conditions.
Full-text · Article · Mar 2013 · Molecular and Cellular Biology