CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications.

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.

Epigenetic Regulation of Endothelial-to-Mesenchymal Transition in Chronic Heart Disease.

Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose their properties and transform into fibroblast-like cells. This transition process contributes to cardiac fibrosis, a common feature of patients with chronic heart failure. To date, no specific therapies to halt or reverse cardiac fibrosis are available, so knowledge of the underlying mechanisms of cardiac fibrosis is urgently needed. In addition, EndMT contributes to other cardiovascular pathologies such as atherosclerosis and pulmonary hypertension, but also to cancer and organ fibrosis. Remarkably, the molecular mechanisms driving EndMT are largely unknown. Epigenetics play an important role in regulating gene transcription and translation and have been implicated in the EndMT process. Therefore, epigenetics might be the missing link in unraveling the underlying mechanisms of EndMT. Here, we review the involvement of epigenetic regulators during EndMT in the context of cardiac fibrosis. The role of DNA methylation, histone modifications (acetylation and methylation), and noncoding RNAs (microRNAs, long noncoding RNAs, and circular RNAs) in the facilitation and inhibition of EndMT are discussed, and potential therapeutic epigenetic targets will be highlighted.

Loss of tet methyl cytosine dioxygenase 3 (TET3) enhances cardiac fibrosis via modulating the DNA damage repair response.

BACKGROUND: Cardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential. RESULT: In this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-ß to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3's recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-ß-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration. CONCLUSION: Collectively, our findings demonstrate TET3's pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.

Cost-effectiveness and cost-utility of optimized mercaptopurine treatment versus placebo in ulcerative colitis patients: The randomized controlled OPTIC trial.

BACKGROUND AND AIMS: We assessed the cost-effectiveness and cost-utility of therapeutic drug monitoring (TDM)-guided mercaptopurine treatment compared with placebo in ulcerative colitis (UC) patients failing 5-aminosalicylates. METHODS: Data were gathered alongside the randomized controlled OPTIC trial (EudraCT: 2015-005260-41). The evaluation was performed from a health care and societal perspective as cost-effectiveness and cost-utility analyses with a time horizon of one year. Volumes and costs of in-hospital care, out-of-hospital care, out-of-pocket expenses and productivity loss were assessed. The main outcomes were the extra costs per additional patient who achieved clinical remission and endoscopic improvement at 52 weeks (responders) and extra costs per quality-adjusted life-year (QALY) gained. RESULTS: In total, 59 patients were randomized to the intervention (n = 29) and control (n = 30) group. Non-significant differences in costs were €63 (-€1267 to €1434; P = 0.93) in favour of placebo from a health care perspective and -€742 (-€3683 to €2016; P = 0.64) in favour of mercaptopurine from a societal perspective. The higher proportion of responders and a non-significant QALY difference of 0.0475 (-0.024-0.117) (P = 0.184) favouring patients on mercaptopurine treatment resulted in €165 extra costs per additional responder and €1326 extra costs per QALY gained from a health care perspective. From a societal perspective, dominance over placebo was observed with cost savings of €1937 per additional responder and €15,621 per QALY gained. The probability of optimised mercaptopurine treatment being cost-effective was 0.80 at a willingness to pay per additional QALY of €20,000. CONCLUSIONS: TDM-based mercaptopurine treatment in UC patients failing 5-aminosalicylates is a cost-effective strategy from a societal perspective.

Efficacious dosing regimens for anti-TNF therapies in inflammatory bowel disease: where do we stand?

INTRODUCTION: During the last decades, biologics have revolutionized the treatment of Crohn's disease and ulcerative colitis. Even though the inflammatory bowel disease (IBD) armamentarium is rapidly expanding with novel biologics, anti-tumor necrosis factor (TNF) antibodies remain the first-line biologic therapy in most areas of the world. However, anti-TNF therapy is not effective in all patients (primary non-response) and patients can lose effect over time (secondary loss of response). AREAS COVERED: This review provides an overview of the current induction and maintenance dosing regimens of the available anti-TNF antibodies and associated challenges in adult patients with IBD. We outline different strategies to overcome these difficulties, including combination therapy, therapeutic drug monitoring (TDM), and dose escalation. Finally, we discuss expected future progress in anti-TNF management. EXPERT OPINION: Anti-TNF agents will remain a cornerstone of IBD treatment in the coming decade. Progress will be made in biomarkers for the prediction of response and individualized dosing regimens. The advent of subcutaneous infliximab challenges the need for concomitant immunosuppression.

miR-132-3p and KLF7 as novel regulators of aortic stiffening-associated EndMT in type 2 diabetes mellitus.

BACKGROUND: The prevalence of diabetes mellitus has risen considerably and currently affects more than 422 million people worldwide. Cardiovascular diseases including myocardial infarction and heart failure represent the major cause of death in type 2 diabetes (T2D). Diabetes patients exhibit accelerated aortic stiffening which is an independent predictor of cardiovascular disease and mortality. We recently showed that aortic stiffness precedes hypertension in a mouse model of diabetes (db/db mice), making aortic stiffness an early contributor to cardiovascular disease development. Elucidating how aortic stiffening develops is a pressing need in order to halt the pathophysiological process at an early time point. METHODS: To assess EndMT occurrence, we performed co-immunofluorescence staining of an endothelial marker (CD31) with mesenchymal markers (α-SMA/S100A4) in aortic sections from db/db mice. Moreover, we performed qRT-PCR to analyze mRNA expression of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. To identify the underlying mechanism by which EndMT contributes to aortic stiffening, we used aortas from db/db mice and diabetic patients in combination with high glucose-treated human umbilical vein endothelial cells (HUVECs) as an in vitro model of diabetes-associated EndMT. RESULTS: We demonstrate robust CD31/α-SMA and CD31/S100A4 co-localization in aortic sections of db/db mice which was almost absent in control mice. Moreover, we demonstrate a significant upregulation of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. As underlying regulator, we identified miR-132-3p as the most significantly downregulated miR in the micronome of db/db mice and high glucose-treated HUVECs. Indeed, miR-132-3p was also significantly downregulated in aortic tissue from diabetic patients. We identified Kruppel-like factor 7 (KLF7) as a target of miR-132-3p and show a significant upregulation of KLF7 in aortic sections of db/db mice and diabetic patients as well as in high glucose-treated HUVECs. We further demonstrate that miR-132-3p overexpression and KLF7 downregulation ameliorates EndMT in high glucose-treated HUVECs. CONCLUSIONS: We demonstrate for the first time that EndMT contributes to aortic stiffening in T2D. We identified miR-132-3p and KLF7 as novel EndMT regulators in this context. Altogether, this gives us new insights in the development of aortic stiffening in T2D.

Early versus Late Use of Vedolizumab in Ulcerative Colitis: Clinical, Endoscopic, and Histological Outcomes.

BACKGROUND AND AIMS: We explored the potential for differential efficacy of vedolizumab between "early" and "late" ulcerative colitis (UC) with evaluation of clinical, endoscopic, and histological endpoints. METHODS: This was a multicentre, multinational open-label study in patients with moderately-to-severely active UC, defining "early" UC by a disease duration <4 years and bio-naïve and "late" UC by a disease duration >4 years and additional exposure to tumour necrosis factor antagonists. Patients received standard treatment with intravenous vedolizumab for 52 weeks (300 mg weeks 0-2-6, every 8 weeks thereafter without escalation). The primary endpoint was corticosteroid-free clinical remission with endoscopic improvement (total Mayo score ≤2 with no subscore >1) at both week 26 and 52. RESULTS: A total of 121 patients were included: in the "early" group 25/59 (42.4%) achieved the primary endpoint versus 19/62 (30.6%) in the "late" group (P = 0.18). There were no significant differences between the two groups in endoscopic improvement (week 26: "early" 32/59 [54.2%] vs. "late" 29/62 [46.8%]; P = 0.412; week 52: 27/59 [45.8%] vs. 25/62 [40.3%]; P = 0.546) or histological remission (Robarts Histopathology Index <3 without neutrophils in the epithelium and lamina propria) (week 26: 24/59 [40.7%] vs. 21/62 [33.9%]; P = 0.439; week 52: 22/59 [37.3%] vs. 22/62 [35.5%]; P = 0.837). CONCLUSIONS: No significant differences in clinical, endoscopic, and histological outcomes were observed between "early" and "late" disease.

Emerging therapies for ulcerative colitis.

INTRODUCTION: Despite advances in the medical management of ulcerative colitis (UC), a subgroup of patients does not respond to currently available therapies. A number of novel drugs are in late stages of clinical development or have recently received regulatory approval for UC. AREAS COVERED: This review focuses on three drug classes that have recently been approved or are awaiting approval for UC: antibodies against interleukin (IL)-23, sphingosine-1-phosphate receptor (S1PR) modulators, and selective inhibitors of Janus kinases (JAK). We provide an overview of their mechanism of action and summarize available evidence for their efficacy and safety. Finally, we discuss expected future challenges in UC management. EXPERT OPINION: The evaluated drugs have demonstrated efficacy with an acceptable safety profile. IL-23 antagonists appear to be safest with very few (serious) adverse events, while the use of S1PR modulators or JAK inhibitors has been associated with infectious and cardiovascular/thromboembolic events, albeit in low numbers. Although advances in drug development are promising, there is an urgent need for (validated) biomarkers to guide rational treatment selection. The scarcity of head-to-head trials also complicates comparisons between available drugs. Breaking the therapeutic ceiling of efficacy in UC will require marked advances in management extending well beyond drug development.

Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1.

Rationale: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure. Methods: We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. In vitro TGFß1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the Rxfp1 promoter region in MCECs. Results: Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFß-pSMAD2/3 signaling in endothelial cells. Conclusions: This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.

Non-coding RNA in endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) is the process wherein endothelial cells lose their typical endothelial cell markers and functions and adopt a mesenchymal-like phenotype. EndMT is required for development of the cardiac valves, the pulmonary and dorsal aorta, and arterial maturation, but activation of the EndMT programme during adulthood is believed to contribute to several pathologies including organ fibrosis, cardiovascular disease, and cancer. Non-coding RNAs, including microRNAs, long non-coding RNAs, and circular RNAs, modulate EndMT during development and disease. Here, we review the mechanisms by which non-coding RNAs facilitate or inhibit EndMT during development and disease and provide a perspective on the therapeutic application of non-coding RNAs to treat fibroproliferative cardiovascular disease.

Causal Connections From Chronic Kidney Disease to Cardiac Fibrosis.

Cardiovascular disease and heart failure are the primary cause of morbidity and mortality in patients with chronic kidney disease. Because impairment of kidney function correlates with heart failure and cardiac fibrosis, a kidney-heart axis is suspected. Although our understanding of the underlying mechanisms still is evolving, the possibility that kidney-heart messengers could be intercepted offers ample reason to focus on this clinically highly relevant problem. Here, we review the current knowledge of how kidney injury causes heart failure and fibrosis.

High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis.

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.

Hypoxia-induced endothelial-mesenchymal transition is associated with RASAL1 promoter hypermethylation in human coronary endothelial cells.

Cardiac fibrosis is integral in chronic heart disease, and one of the cellular processes contributing to cardiac fibrosis is endothelial-to-mesenchymal transition (EndMT). We recently found that hypoxia efficiently induces human coronary artery endothelial cells (HCAEC) to undergo EndMT through a hypoxia inducible factor-1α (HIF1α)-dependent pathway. Promoter hypermethylation of Ras-Gap-like protein 1 (RASAL1) has also been recently associated with EndMT progression and cardiac fibrosis. Our findings suggest that HIF1α and transforming growth factor (TGF)/SMAD signalling pathways synergistically regulate hypoxia-induced EndMT through both DNMT3a-mediated hypermethylation of RASAL1 promoter and direct SNAIL induction. The findings indicate that multiple cascades may be activated simultaneously to mediate hypoxia-induced EndMT.