Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.

Adverse Impact of Cannabis on Human Health.

Cannabis, the most commonly used recreational drug, is illicit in many areas of the world. With increasing decriminalization and legalization, cannabis use is increasing in the United States and other countries. The adverse effects of cannabis are unclear because its status as a Schedule 1 drug in the United States restricts research. Despite a paucity of data, cannabis is commonly perceived as a benign or even beneficial drug. However, recent studies show that cannabis has adverse cardiovascular and pulmonary effects and is linked with malignancy. Moreover, case reports have shown an association between cannabis use and neuropsychiatric disorders. With growing availability, cannabis misuse by minors has led to increasing incidences of overdose and toxicity. Though difficult to detect, cannabis intoxication may be linked to impaired driving and motor vehicle accidents. Overall, cannabis use is on the rise, and adverse effects are becoming apparent in clinical data sets.

Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.

Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.

MiR-33a is a therapeutic target in SPG4-related hereditary spastic paraplegia human neurons.

Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3'-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.

MicroRNA-33 Controls Adaptive Fibrotic Response in the Remodeling Heart by Preserving Lipid Raft Cholesterol.

RATIONALE: Heart failure and atherosclerosis share the underlying mechanisms of chronic inflammation followed by fibrosis. A highly conserved microRNA (miR), miR-33, is considered as a potential therapeutic target for atherosclerosis because it regulates lipid metabolism and inflammation. However, the role of miR-33 in heart failure remains to be elucidated. OBJECTIVE: To clarify the role of miR-33 involved in heart failure. METHODS AND RESULTS: We first investigated the expression levels of miR-33a/b in human cardiac tissue samples with dilated cardiomyopathy. Increased expression of miR-33a was associated with improving hemodynamic parameters. To clarify the role of miR-33 in remodeling hearts, we investigated the responses to pressure overload by transverse aortic constriction in miR-33-deficient (knockout [KO]) mice. When mice were subjected to transverse aortic constriction, miR-33 expression levels were significantly upregulated in wild-type left ventricles. There was no difference in hypertrophic responses between wild-type and miR-33KO hearts, whereas cardiac fibrosis was ameliorated in miR-33KO hearts compared with wild-type hearts. Despite the ameliorated cardiac fibrosis, miR-33KO mice showed impaired systolic function after transverse aortic constriction. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart. Deficiency of miR-33 impaired cardiac fibroblast proliferation, which was considered to be caused by altered lipid raft cholesterol content. Moreover, cardiac fibroblast-specific miR-33-deficient mice also showed decreased cardiac fibrosis induced by transverse aortic constriction as systemic miR-33KO mice. CONCLUSION: Our results demonstrate that miR-33 is involved in cardiac remodeling, and it preserves lipid raft cholesterol content in fibroblasts and maintains adaptive fibrotic responses in the remodeling heart.

SREBF1/MicroRNA-33b Axis Exhibits Potent Effect on Unstable Atherosclerotic Plaque Formation In Vivo.

Objective- Atherosclerosis is a common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects; however, the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo because of lack of miR-33b sequences in mice, unlike human. Approach and Results- Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under apolipoprotein E-deficient background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum high-density lipoprotein cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions- Our data demonstrated critical roles of SREBF1-miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.

Genetic Ablation of MicroRNA-33 Attenuates Inflammation and Abdominal Aortic Aneurysm Formation via Several Anti-Inflammatory Pathways.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is an increasingly prevalent and ultimately fatal disease with no effective pharmacological treatment. Because matrix degradation induced by vascular inflammation is the major pathophysiology of AAA, attenuation of this inflammation may improve its outcome. Previous studies suggested that miR-33 (microRNA-33) inhibition and genetic ablation of miR-33 increased serum high-density lipoprotein cholesterol and attenuated atherosclerosis. APPROACH AND RESULTS: MiR-33a-5p expression in central zone of human AAA was higher than marginal zone. MiR-33 deletion attenuated AAA formation in both mouse models of angiotensin II- and calcium chloride-induced AAA. Reduced macrophage accumulation and monocyte chemotactic protein-1 expression were observed in calcium chloride-induced AAA walls in miR-33-/- mice. In vitro experiments revealed that peritoneal macrophages from miR-33-/- mice showed reduced matrix metalloproteinase 9 expression levels via c-Jun N-terminal kinase inactivation. Primary aortic vascular smooth muscle cells from miR-33-/- mice showed reduced monocyte chemotactic protein-1 expression by p38 mitogen-activated protein kinase attenuation. Both of the inactivation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were possibly because of the increase of ATP-binding cassette transporter A1 that is a well-known target of miR-33. Moreover, high-density lipoprotein cholesterol derived from miR-33-/- mice reduced expression of matrix metalloproteinase 9 in macrophages and monocyte chemotactic protein-1 in vascular smooth muscle cells. Bone marrow transplantation experiments indicated that miR-33-deficient bone marrow cells ameliorated AAA formation in wild-type recipients. MiR-33 deficiency in recipient mice was also shown to contribute the inhibition of AAA formation. CONCLUSIONS: These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.

MicroRNA-451 exacerbates lipotoxicity in cardiac myocytes and high-fat diet-induced cardiac hypertrophy in mice through suppression of the LKB1/AMPK pathway.

RATIONALE: In some patients with type 2 diabetes mellitus (DM) without hypertension, cardiac hypertrophy and attenuated cardiac function are observed, and this insult is termed diabetic cardiomyopathy. To date, microRNA (miRNAs or miR) functions in diabetic cardiomyopathy remain to be elucidated. OBJECTIVE: To clarify the functions of miRNAs involved in diabetic cardiomyopathy caused by type 2 DM. METHODS AND RESULTS: C57BL/6 mice were fed a high-fat diet (HFD) for 20 weeks, which induced obesity and type 2 DM. miRNA microarray analyses and real-time polymerase chain reaction revealed that miR-451 levels were significantly increased in the type 2 DM mouse hearts. Because excess supply of saturated fatty acids is a cause of diabetic cardiomyopathy, we stimulated neonatal rat cardiac myocytes with palmitic acid and confirmed that miR-451 expression was increased in a dose- and time-dependent manner. Loss of miR-451 function ameliorated palmitate-induced lipotoxicity in neonatal rat cardiac myocytes. Calcium-binding protein 39 (Cab39) is a scaffold protein of liver kinase B1 (LKB1), an upstream kinase of AMP-activated protein kinase (AMPK). Cab39 was a direct target of miR-451 in neonatal rat cardiac myocytes and Cab39 overexpression rescued the lipotoxicity. To clarify miR-451 functions in vivo, we generated cardiomyocyte-specific miR-451 knockout mice. HFD-induced cardiac hypertrophy and contractile reserves were ameliorated in cardiomyocyte-specific miR-451 knockout mice compared with control mice. Protein levels of Cab39 and phosphorylated AMPK were increased and phosphorylated mammalian target of rapamycin (mTOR) was reduced in cardiomyocyte-specific miR-451 knockout mouse hearts compared with control mouse hearts. CONCLUSIONS: Our results demonstrate that miR-451 is involved in diabetic cardiomyopathy through suppression of the LKB1/AMPK pathway.

Prognostic Significance of ST-Segment Elevation in Leads V1-2 in Patients With Severe Aortic Stenosis.

BACKGROUND: ST-segment elevation (STE) in leads V1-2 is often observed in patients with severe aortic stenosis (AS), but its significance remains unknown. METHODS AND RESULTS: We retrospectively evaluated baseline ECGs and 5-year clinical outcomes in 211 consecutive patients with severe AS, defined as peak aortic jet velocity (Aortic Vmax) >4.0 m/s, or mean aortic pressure gradient >40 mmHg, or aortic valve area (AVA) <1.0 cm(2). The primary outcome measure was a composite of death or surgical aortic valve replacement (AVR). Patients with STE in leads V1-2(≥0.15 mV) had greater Aortic Vmax and smaller AVA than patients without. With a median follow-up of 4.9 years, the cumulative 5-year incidence of death or AVR was significantly higher in patients with STE in leads V1-2 than in patients without (91.4% vs. 77.1%; P=0.003). After adjusting for confounders, STE in leads V1-2 was independently associated with higher risk for death or AVR (hazard ratio, 1.53; 95% confidence interval, 1.06-2.22; P=0.02). In 64 asymptomatic patients without any indication for AVR at initial diagnosis of severe AS, the cumulative incidence of AVR was significantly higher in patients with STE in leads V1-2 than in patients without (57.6% vs. 30.5%; P<0.001). CONCLUSIONS: STE in leads V1-2 independently predicted poorer prognosis and more frequent need for AVR in patients with severe AS.

Predictors of Rapid Progression and Clinical Outcome of Asymptomatic Severe Aortic Stenosis.

BACKGROUND: The optimal timing of aortic valve replacement (AVR) is controversial in patients with asymptomatic severe aortic stenosis (AS) except when very severe. Prediction of progression of severe AS is helpful in deciding on the timing of AVR. The purpose of this study was to clarify the predictors of progression rate and clinical outcomes of severe AS. METHODS AND RESULTS: We retrospectively investigated 140 consecutive patients with asymptomatic severe AS (aortic valve area [AVA], 0.75-1.0 cm(2)). First-year progression rate and annual progression rate of AVA and of aortic jet velocity (AV-Vel) were calculated. Cardiac events were examined and the predictors of rapid progression and cardiac events were analyzed. The median follow-up period was 36 months. The median annual progression rate was -0.05 cm(2)/year for AVA and 0.22 m/s/year for AV-Vel. Dyslipidemia, moderate-severe calcification, and first-year AV-Vel progression ≥0.22 m/s/year were independent predictors of cardiac events. Cardiac event-free rate was lower in patients with AV-Vel first-year progression rate ≥0.22 m/s/year than in those with a lower rate. Diabetes and moderate-severe calcification were related to first-year rapid progression. CONCLUSIONS: The annual progression rate of severe AS was -0.05 cm(2)/year for AVA and 0.22 m/s/year for AV-Vel. Patients with first-year rapid progression or severely calcified aortic valve should be carefully observed while considering an early operation. (Circ J 2016; 80: 1863-1869).

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context.

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

The use of new CRISPR tools in cardiovascular research and medicine.

Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.

Progression of isolated tricuspid regurgitation late after left-sided valve surgery. Clinical features and mechanisms.

BACKGROUND: Severe tricuspid regurgitation (TR) sometimes develops late after left-sided valve surgery without left heart failure, pulmonary hypertension or rheumatic tricuspid valve. The purpose of the present study was to investigate clinical characteristics and mechanisms of severe isolated TR late after left-sided valve surgery. METHODS AND RESULTS: A total of 372 consecutive patients who underwent left-sided valve surgery between 1990 and 2003 and who were followed up with echocardiography for at least 5 years, were retrospectively investigated. The mean follow-up period was 9.4 years. Clinical background, preoperative and postoperative echocardiographic parameters were evaluated. Among the 372 patients, severe isolated TR was detected in 23 patients, which developed at a mean of 8.6 years after surgery. Twenty-two of 23 patients had undergone mitral valve surgery. Multivariate logistic regression analysis identified the presence of preoperative atrial fibrillation and preoperative ejection fraction as independent determinants for the development of severe isolated TR. In patients with severe isolated TR, the tricuspid annular diameter and the right atrial area were already enlarged early after surgery and both of these increased prior to TR progression. CONCLUSIONS: Severe isolated TR developing late after mitral valve surgery is not uncommon, thus it is important to recognize this disease entity. Annular dilatation was the main cause of isolated TR and serial echocardiographic data are important to detect progression of isolated TR and to assess its mechanisms.

CRISPRi/a Screening with Human iPSCs.

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.

Therapeutic genome editing in cardiovascular diseases.

During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.

microRNA-33 maintains adaptive thermogenesis via enhanced sympathetic nerve activity.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33-/- mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-ß-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.