Kemal Marc Akat

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers

Significance Heart failure (HF) has a high morbidity and mortality and its incidence is increasing worldwide. While protein biomarkers have been established for diagnostic and prognostic evaluation of patients with HF, there is currently no systematic assessment of RNA biomarkers. We determined the composition of myocardial tissue and circulating microRNAs (miRNAs) in a large cohort of patients with stable and advanced HF and compared it to the composition of normal adult and fetal samples. The advanced HF patients underwent mechanical unloading with left ventricular assist devices and samples were collected at different postoperative time points. Our findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers. Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA–target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.

Kruppel-like Factor 15 Is a Critical Regulator of Cardiac Lipid Metabolism

Background: Metabolic homeostasis is central to normal cardiac function. The molecular mechanisms underlying metabolic plasticity in the heart remain poorly understood. Results: Kruppel-like factor 15 (KLF15) is a direct and independent regulator of myocardial lipid flux. Conclusion: KLF15 is a core component of the transcriptional circuitry that governs cardiac metabolism. Significance: This work is the first to implicate the KLF transcription factor family in cardiac metabolism. The mammalian heart, the bodys largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.

Single cell RNA sequencing to dissect the molecular heterogeneity in lupus nephritis

Lupus nephritis is a leading cause of mortality among systemic lupus erythematosus (SLE) patients, and its heterogeneous nature poses a significant challenge to the development of effective diagnostics and treatments. Single cell RNA sequencing (scRNA-seq) offers a potential solution to dissect the heterogeneity of the disease and enables the study of similar cell types distant from the site of renal injury to identify novel biomarkers. We applied scRNA-seq to human renal and skin biopsy tissues and demonstrated that scRNA-seq can be performed on samples obtained during routine care. Chronicity index, IgG deposition, and quantity of proteinuria correlated with a transcriptomic-based score composed of IFN-inducible genes in renal tubular cells. Furthermore, analysis of cumulative expression profiles of single cell keratinocytes dissociated from nonlesional, non-sun-exposed skin of patients with lupus nephritis also revealed upregulation of IFN-inducible genes compared with keratinocytes isolated from healthy controls. This indicates the possible use of scRNA-seq analysis of skin biopsies as a biomarker of renal disease. These data support the potential utility of scRNA-seq to provide new insights into the pathogenesis of lupus nephritis and pave the way for exploiting a readily accessible tissue to reflect injury in the kidney.

Unique microRNAs appear at different times during the course of a delayed-type hypersensitivity reaction in human skin

Diphencyprone (DPCP) is a hapten that induces delayed‐type hypersensitivity (DTH) reactions. MicroRNAs (miRNAs) are short non‐coding RNAs that negatively regulate gene expression and have been implicated in various inflammatory skin diseases, but their role in DTH reactions is not well understood. We generated global miRNA expression profiles (using next‐generation sequencing) of DPCP reactions in skin of seven healthy volunteers at 3, 14 and 120 days after challenge. Compared to placebo‐treated sites, DPCP‐challenged skin at 3 days (peak inflammation) had 127 miRNAs significantly deregulated. At 14 days (during resolution of inflammation), 43 miRNAs were deregulated and, at 120 days (when inflammation had completely resolved), six miRNAs were upregulated. While some miRNAs have been observed in psoriasis or atopic dermatitis, most of the deregulated miRNAs have not yet been studied in the context of skin biology or immunology. Across the three time points studied, many but not all miRNAs were uniquely expressed. As various miRNAs may influence T cell activation, this may indicate that the miRNAs exclusively expressed at different time points function to promote or resolve skin inflammation, and therefore, may inform on the paradoxical ability of DPCP to treat both autoimmune conditions (alopecia areata) and conditions of ineffective immunity (melanoma).

Human plasma and serum extracellular small RNA reference profiles and their clinical utility

Significance Nucleic acids mediate storage and expression of genetic information. Extracellular DNA (exDNA) and exRNA are traces of nucleic acids released from cells into the extracellular environment. Their use as disease biomarkers has been limited by technical challenges in their isolation caused by abundant RNA- and DNA-degrading enzymes in biofluids. Using isolation protocols developed especially for biofluids, we generated plasma and serum exRNA reference profiles from 13 healthy volunteers over time and determined the effect of critical clinical parameters such as gender and fasting. Surprisingly, we encountered one participant with dramatically increased endocrine-origin exRNA contributions stable over 1 year and detectable in all of his samples, thereby demonstrating the robustness of this approach and the clinical potential of circulating RNAs as biomarkers. Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell–specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell–specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.

Deciphering human ribonucleoprotein regulatory networks

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules encoded functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.

Modulation of LIN28B/Let-7 Signaling by Propranolol Contributes to Infantile Hemangioma Involution

Objective— Infantile hemangiomas (IHs) are the most common benign vascular neoplasms of infancy, characterized by a rapid growth phase followed by a spontaneous involution, or triggered by propranolol treatment by poorly understood mechanisms. LIN28/let-7 axis plays a central role in the regulation of stem cell self-renewal and tumorigenesis. However, the role of LIN28B/let-7 signaling in IH pathogenesis has not yet been elucidated. Approach and Results— LIN28B is highly expressed in proliferative IH and is less expressed in involuted and in propranolol-treated IH samples as measured by immunofluorescence staining and quantitative RT-PCR. Small RNA sequencing analysis of IH samples revealed a decrease in microRNAs that target LIN28B, including let-7, and an increase in microRNAs in the mir-498(46) cistron. Overexpression of LIN28B in HEK293 cells induced the expression of miR-516b in the mir-498(46) cistron. Propranolol treatment of induced pluripotent stem cells, which express mir-498(46) endogenously, reduced the expression of both LIN28B and mir-498(46) and increased the expression of let-7. Furthermore, propranolol treatment reduced the proliferation of induced pluripotent stem cells and induced epithelial–mesenchymal transition. Conclusions— This work uncovers the role of the LIN28B/let-7 switch in IH pathogenesis and provides a novel mechanism by which propranolol induces IH involution. Furthermore, it provides therapeutic implications for cancers in which the LIN28/let-7 pathway is imbalanced.