Victoria N. Parikh

MicroRNA-21 Integrates Pathogenic Signaling to Control Pulmonary Hypertension Results of a Network Bioinformatics Approach

Background— Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA molecules are potential candidates for coordinated regulation of these disease stimuli. Methods and Results— Using a network biology approach, we identify microRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying microRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho-kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haploinsufficiency of BMP receptor type 2. To validate these predictions, we have found that hypoxia and BMP receptor type 2 signaling independently upregulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 downregulates BMP receptor type 2 expression. Furthermore, miR-21 directly represses RhoB expression and Rho-kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is upregulated in pulmonary tissue from several rodent models of PH and in humans with PH. On induction of disease in miR-21–null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. Conclusions— A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying microRNA in PH.Background— Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA molecules are potential candidates for coordinated regulation of these disease stimuli. Methods and Results— Using a network biology approach, we identify microRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying microRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho-kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haploinsufficiency of BMP receptor type 2. To validate these predictions, we have found that hypoxia and BMP receptor type 2 signaling independently upregulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 downregulates BMP receptor type 2 expression. Furthermore, miR-21 directly represses RhoB expression and Rho-kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is upregulated in pulmonary tissue from several rodent models of PH and in humans with PH. On induction of disease in miR-21 –null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. Conclusions— A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying microRNA in PH. # Clinical Perspective {#article-title-52}

Androgen level and male social status in the African cichlid, Astatotilapia burtoni.

In vertebrates, circulating androgen levels are regulated by the hypothalamic-pituitary-gonadal (HPG) axis through which the brain controls the gonads via the pituitary. Androgen levels ultimately depend on factors including season, temperature, social circumstance, age, and other variables related to reproductive capacity and opportunity. Previous studies with an African cichlid fish, Astatotilapia burtoni, suggested that changes in both testosterone and 11-ketotestosterone (11-KT), an androgen specific to teleost fish, depend on male social status. Here we characterize circulating plasma concentrations of testosterone and 11-KT in socially dominant (territorial) and socially subordinate (non-territorial) males. Territorial males have significantly higher circulating levels of both forms of androgen, which is another defining difference between dominant and subordinate males in this species. These results underscore how internal and external cues related to reproduction are integrated at the level of the HPG axis.

Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension

Dysregulation of vascular stiffness and cellular metabolism occurs early in pulmonary hypertension (PH). However, the mechanisms by which biophysical properties of the vascular extracellular matrix (ECM) relate to metabolic processes important in PH remain undefined. In this work, we examined cultured pulmonary vascular cells and various types of PH-diseased lung tissue and determined that ECM stiffening resulted in mechanoactivation of the transcriptional coactivators YAP and TAZ (WWTR1). YAP/TAZ activation modulated metabolic enzymes, including glutaminase (GLS1), to coordinate glutaminolysis and glycolysis. Glutaminolysis, an anaplerotic pathway, replenished aspartate for anabolic biosynthesis, which was critical for sustaining proliferation and migration within stiff ECM. In vitro, GLS1 inhibition blocked aspartate production and reprogrammed cellular proliferation pathways, while application of aspartate restored proliferation. In the monocrotaline rat model of PH, pharmacologic modulation of pulmonary vascular stiffness and YAP-dependent mechanotransduction altered glutaminolysis, pulmonary vascular proliferation, and manifestations of PH. Additionally, pharmacologic targeting of GLS1 in this model ameliorated disease progression. Notably, evaluation of simian immunodeficiency virus-infected nonhuman primates and HIV-infected subjects revealed a correlation between YAP/TAZ-GLS activation and PH. These results indicate that ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis and anaplerosis, and thereby link mechanical stimuli to dysregulated vascular metabolism. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in PH.

Behavioral coping strategies in a cichlid fish: the role of social status and acute stress response in direct and displaced aggression.

The African cichlid fish, Astatotilapia burtoni, has a complex social system with a sophisticated social hierarchy that offers unique opportunities to understand how social rank and its physiological substrates relate to behavioral strategies. In A. burtoni, a small fraction of the males are dominant (T, territorial), as distinguished by being large, brightly colored, reproductively active, and aggressively defending territories. In contrast, the majority of males are non-dominant (NT, non-territorial), being smaller, drably colored, sexually immature, and typically schooling with females. The social system is regulated by aggressive interactions between males and behavioral responses to aggression can be direct or displaced with respect to the animal that acts. To determine whether direct and displaced behaviors are differentially exhibited by T and NT males, individuals were shown a video presentation of a dominant male displaying aggressively. Analysis of aggressive acts toward the video display and displaced activity toward a tank mate revealed that T males exhibited more direct behavior (toward the video display), while NT males engaged in more displaced behavior (toward tank mates). Because similar experiments with primates suggest that shifts in behavioral strategies are linked to changes in the stress response (as measured by circulating cortisol levels), we measured cortisol levels of T and NT males following exposure to the aggressive stimulus. Although in some animals subordinate males are reported to have higher cortisol levels, here we show that in A. burtoni the endocrine response to specific situations can vary considerably even among animals of the same status. Interestingly, NT males with intermediate cortisol levels showed more directed behavior while NT males with both high and low cortisol levels showed more displaced. This suggests an optimal physiological stress response in NT males that predisposes them to challenge aggressors perhaps making it more likely for them to ascend in status.

Next-Generation Sequencing in Cardiovascular Disease: Present Clinical Applications and the Horizon of Precision Medicine.

The initial sequencing of the human genome took nearly a decade to complete. Today, the same can be accomplished in hours using next-generation sequencing (NGS). Before the invention of NGS, genetic testing was accomplished by traditional Sanger sequencing. In cardiovascular medicine, this approach typically involved the sequencing of a region of interest in a single affected patient’s DNA and then comparison of that sequence to the human genome reference sequence. Variants found were then compared to a panel of sequences from unaffected people, often anonymous blood donors. This strategy has been the mainstay of genetic testing to assess small numbers of disease-related genes across cardiovascular and noncardiovascular diseases. However, this approach is limited in its scalability. NGS builds on these basic principles, extending them to allow billions of DNA molecules to be sequenced simultaneously. This multiplex technology yields billions of small polynucleotide reads, called “short reads.” Mapping and assembly of these short reads to a reference genome then allows discovery of genetic variants at scale (Figure 1), increasing the speed and reducing the cost of sequencing by orders of magnitude.1 Thus, NGS can easily be applied to panels of genes, every gene, or the whole genome in clinical settings. This scientific leap in sequencing technology has occurred over just 15 years and has brought the genetic code to the forefront of diagnosis and therapy, making it increasingly important for clinicians to be aware of its utility and limitations in practice. Figure 1. Comparison of traditional sequencing and next-generation sequencing (NGS). In traditional sequencing, DNA is replicated in the presence of fluorescently labeled bases, yielding differently sized strands with different terminal bases. These terminal bases are identified to assemble the entire sequence and compare it …

Brief Report: Coordinated Modulation of Circulating miR-21 in HIV, HIV-Associated Pulmonary Arterial Hypertension, and HIV/Hepatitis C Virus Coinfection.

Abstract:Dysregulation of microRNA-21 (miR-21) is independently associated with HIV infection, pulmonary arterial hypertension (PAH), and hepatitis C virus (HCV) infection. To assess the expression of miR-21 in these overlapping comorbidities, we measured plasma miR-21 in HIV with and without PAH and then stratified by concomitant HCV infection. MiR-21 was increased in HIV and HIV-PAH versus uninfected subjects, but it did not differ between these groups. HIV/HCV coinfection correlated with even higher miR-21 levels within the HIV-infected population. These data reveal specific regulation of plasma miR-21 in HIV, HIV/HCV coinfection, and PAH and suggest that miR-21 may integrate complex disease-specific signaling in the setting of HIV infection.

Delivering Clinical Grade Sequencing and Genetic Test Interpretation for Cardiovascular Medicine

Contemporary DNA sequencing approaches are increasingly used as diagnostic tools within clinical medicine, driven by rapid reductions in cost and improvements in speed. In 2014, Illumina, Inc. launched a system that could sequence an entire human genome for under

Mind the Gap: Current Challenges and Future State of Heart Failure Care

1000,1 with sequencing and analysis achievable in under 2 days.2,3 For inherited disease, next-generation sequencing (NGS) technologies have been applied in 3 broad categories: (1) gene panels, where a collection of predefined genes for a given condition, or a group of closely related conditions, are sequenced; (2) whole-exome sequencing (WES), where the majority of the protein-coding portions of the genome (≈2% of the genome) is sequenced; and (3) whole-genome sequencing (WGS), where the majority of the genome is sequenced, including nonprotein-coding DNA. In the management of inherited cardiovascular disease, there has been increasing use of genetic testing as major healthcare systems establish centers of excellence. In most cases, these tests now feature NGS approaches. However, as we transition from traditional to NGS approaches, it is important that noninferiority with traditional practices is firmly established. Furthermore, our ability to correctly interpret the clinical impact of variants derived from these sequencing efforts remains suboptimal. The recent analysis and public release of sequence data from tens of thousands of individuals established that rare variation is common in humans, meaning that only a small proportion of rare variants will actually be causal of rare genetic disease. Indeed, even when rare variants emerge that could potentially explain a given presentation, the classification of such variants is often discordant between laboratories. Here, we will explore the challenge and opportunity of NGS for inherited cardiovascular disease. First, we describe recent advances in sequencing and interpretation facilitated by large-scale population genomics studies. Next, we outline current approaches to clinical genetic testing and describe areas of …

Wrestling the Giant: New Approaches for Assessing Titin Variant Pathogenicity

The past decade has seen many advances in the management of heart failure (HF) that have improved survival and quality of life for patients living with this condition. A number of gaps remain in our understanding of the pathophysiology of HF, and the application of emerging treatment strategies is an exciting but daunting challenge. It is possible that advances in genetic evaluation of cardiomyopathy will provide a more refined approach to characterizing HF syndromes, whereas large-scale clinical trials on the horizon should further clarify the role of novel pharmacologic agents and invasive therapies. Cardiac repair and regeneration hold great promise, but a number of pragmatic issues will limit clinical application in the near term. Replacing cardiac function with ventricular assist devices represents significant progress in the management of advanced disease; however, unacceptable rates of complications and costs need to be addressed before broader use in the general HF population is feasible. The ability to personalize care is limited, and the optimal model of disease management in the Canadian context remains uncertain. The emergence of biomarker-guided management and remote monitoring technologies might facilitate a more personalized approach to care in an effort to maintain health and stability and to prevent worsening HF. Ultimately, a greater understanding of how and when to intervene in the setting of acute HF should translate into improved outcomes for the highest-risk subgroup of patients. This review highlights key challenges in the management of HF and highlights the progress toward an ideal future state.

Apelin and APJ orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition

In the recent past, the clinical availability of genetic information has increased exponentially and with it the opportunity to use this information to aid in diagnosis and treatment of cardiovascular disease. One of the greatest challenges we face with this wealth of potentially useful diagnostic information is determining the pathogenicity of the myriad genetic variants identified. Dilated cardiomyopathy (DCM) is a particularly salient example of this challenge. Compared with other inherited cardiovascular diseases, such as long QT syndrome, with a diagnostic yield of genetic testing of ≈80%, only 15% to 40% of DCM genetic tests yield a pathogenic variant.1 This reduced diagnostic capability is due in part to our lack of precision in defining phenotypes, as well as the sheer size and number of genes thought to be involved, rendering the examination of a large number of variants in an individual cumbersome. Articles, see p 419 and 426 In the current issue, 2 groups have addressed an exemplar of these challenges: Titin (TTN ).2,3 TTN encodes the largest human protein, consisting of 364 exons that are spliced into transcripts encoding ≈5000 to 34 000 amino acids. Largely because of its size and the ubiquity of rare variation, many individuals harbor variation in the TTN gene (1%–3% differ from the human reference sequence).4,5 However, significant data support the presence of pathogenic variation in TTN , leading to dilated cardiomyopathies, specifically truncating variants in the A-band. Although consistent cosegregation evidence is lacking,6 such variants clearly have lower prevalence in the healthy population,5,7 highlighting the importance of critical evaluation of clinically observed variation in TTN . In this issue, Hastings et al3 and Deo2 each address this challenge with the use of innovative perspective, proposing novel solutions to the problem of …