The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

OBJECTIVE: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. METHODS: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. RESULTS: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-ß regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. CONCLUSIONS: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility.

Based upon our demonstration that the smooth muscle cell-selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6's role in SMCs in vivo using cell type-specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation-sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

Obesity confers macrophage memory.

Epigenetic programming of myeloid cells in obesity contributes to macular degeneration.

Macrophage-specific inhibition of the histone demethylase JMJD3 decreases STING and pathologic inflammation in diabetic wound repair.

Macrophage plasticity is critical for normal tissue repair following injury. In pathologic states such as diabetes, macrophage plasticity is impaired, and macrophages remain in a persistent proinflammatory state; however, the reasons for this are unknown. Here, using single-cell RNA sequencing of human diabetic wounds, we identified increased JMJD3 in diabetic wound macrophages, resulting in increased inflammatory gene expression. Mechanistically, we report that in wound healing, JMJD3 directs early macrophage-mediated inflammation via JAK1,3/STAT3 signaling. However, in the diabetic state, we found that IL-6, a cytokine increased in diabetic wound tissue at later time points post-injury, regulates JMJD3 expression in diabetic wound macrophages via the JAK1,3/STAT3 pathway and that this late increase in JMJD3 induces NFκB-mediated inflammatory gene transcription in wound macrophages via an H3K27me3 mechanism. Interestingly, RNA sequencing of wound macrophages isolated from mice with JMJD3-deficient myeloid cells (Jmjd3f/fLyz2Cre+) identified that the STING gene (Tmem173) is regulated by JMJD3 in wound macrophages. STING limits inflammatory cytokine production by wound macrophages during healing. However, in diabetic mice, its role changes to limit wound repair and enhance inflammation. This finding is important since STING is associated with chronic inflammation, and we found STING to be elevated in human and murine diabetic wound macrophages at late time points. Finally, we demonstrate that macrophage-specific, nanoparticle inhibition of JMJD3 in diabetic wounds significantly improves diabetic wound repair by decreasing inflammatory cytokines and STING. Taken together, this work highlights the central role of JMJD3 in tissue repair and identifies cell-specific targeting as a viable therapeutic strategy for nonhealing diabetic wounds.

The Role of Epigenetic Modifications in Abdominal Aortic Aneurysm Pathogenesis.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high morbidity and mortality in the setting of acute rupture. Recently, advances in surgical and endovascular repair of AAA have been achieved; however, pharmaceutical therapies to prevent AAA expansion and rupture remain lacking. This highlights an ongoing need to improve the understanding the pathological mechanisms that initiate formation, maintain growth, and promote rupture of AAA. Over the past decade, epigenetic modifications, such as DNA methylation, posttranslational histone modifications, and non-coding RNA, have emerged as important regulators of cellular function. Accumulating studies reveal the importance of epigenetic enzymes in the dynamic regulation of key signaling pathways that alter cellular phenotypes and have emerged as major intracellular players in a wide range of biological processes. In this review, we discuss the roles and implications of epigenetic modifications in AAA animal models and their relevance to human AAA pathology.

Coronavirus induces diabetic macrophage-mediated inflammation via SETDB2.

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.

Palmitate-TLR4 signaling regulates the histone demethylase, JMJD3, in macrophages and impairs diabetic wound healing.

Chronic macrophage inflammation is a hallmark of type 2 diabetes (T2D) and linked to the development of secondary diabetic complications. T2D is characterized by excess concentrations of saturated fatty acids (SFA) that activate innate immune inflammatory responses, however, mechanism(s) by which SFAs control inflammation is unknown. Using monocyte-macrophages isolated from human blood and murine models, we demonstrate that palmitate (C16:0), the most abundant circulating SFA in T2D, increases expression of the histone demethylase, Jmjd3. Upregulation of Jmjd3 results in removal of the repressive histone methylation (H3K27me3) mark on NFκB-mediated inflammatory gene promoters driving macrophage-mediated inflammation. We identify that the effects of palmitate are fatty acid specific, as laurate (C12:0) does not regulate Jmjd3 and the associated inflammatory profile. Further, palmitate-induced Jmjd3 expression is controlled via TLR4/MyD88-dependent signaling mechanism, where genetic depletion of TLR4 (Tlr4-/- ) or MyD88 (MyD88-/- ) negated the palmitate-induced changes in Jmjd3 and downstream NFκB-induced inflammation. Pharmacological inhibition of Jmjd3 using a small molecule inhibitor (GSK-J4) reduced macrophage inflammation and improved diabetic wound healing. Together, we conclude that palmitate contributes to the chronic Jmjd3-mediated activation of macrophages in diabetic peripheral tissue and a histone demethylase inhibitor-based therapy may represent a novel treatment for nonhealing diabetic wounds.

Genetic and epigenetic regulation of abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are focal dilations of the aorta that develop from degenerative changes in the media and adventitia of the vessel. Ruptured AAAs have a mortality of up to 85%, thus it is important to identify patients with AAA at increased risk for rupture who would benefit from increased surveillance and/or surgical repair. Although the exact genetic and epigenetic mechanisms regulating AAA formation are not completely understood, Mendelian cases of AAA, which result from pathologic variants in a single gene, have helped provide a basic understanding of AAA pathophysiology. More recently, genome wide associated studies (GWAS) have identified additional variants, termed single nucleotide polymorphisms, in humans that may be associated with AAAs. While some variants may be associated with AAAs and play causal roles in aneurysm pathogenesis, it should be emphasized that the majority of SNPs do not actually cause disease. In addition to GWAS, other studies have uncovered epigenetic causes of disease that regulate expression of genes known to be important in AAA pathogenesis. This review describes many of these genetic and epigenetic contributors of AAAs, which altogether provide a deeper insight into AAA pathogenesis.

GRAF3 serves as a blood volume-sensitive rheostat to control smooth muscle contractility and blood pressure.

Vascular resistance is a major determinant of BP and is controlled, in large part, by RhoA-dependent smooth muscle cell (SMC) contraction within small peripheral arterioles and previous studies from our lab indicate that GRAF3 is a critical regulator of RhoA in vascular SMC. The elevated contractile responses we observed in GRAF3 deficient vessels coupled with the hypertensive phenotype provided a mechanistic link for the hypertensive locus recently identified within the GRAF3 gene. On the basis of our previous findings that the RhoA signaling axis also controls SMC contractile gene expression and that GRAF3 expression was itself controlled by this pathway, we postulated that GRAF3 serves as an important counter-regulator of SMC phenotype. Indeed, our new findings presented herein indicate that GRAF3 expression acts as a pressure-sensitive rheostat to control vessel tone by both reducing calcium sensitivity and restraining expression of the SMC-specific contractile proteins that support this function. Collectively, these studies highlight the potential therapeutic value of GRAF3 in the control of human hypertension.

Transcriptional and posttranscriptional regulation of the SMC-selective blood pressure-associated gene, ARHGAP42.

We previously showed that ARHGAP42 is a smooth muscle cell (SMC)-selective, RhoA-specific GTPase activating protein that regulates blood pressure and that a minor allele single nucleotide variation within a DNAse hypersensitive regulatory element in intron1 (Int1DHS) increased ARHGAP42 expression by promoting serum response factor binding. The goal of the current study was to identify additional transcriptional and posttranscriptional mechanisms that control ARHGAP42 expression. Using deletion/mutation, gel shift, and chromatin immunoprecipitation experiments, we showed that recombination signal binding protein for immunoglobulin κ-J region (RBPJ) and TEA domain family member 1 (TEAD1) binding to a conserved core region was required for full IntDHS transcriptional activity. Importantly, overexpression of the notch intracellular domain (NICD) or plating SMCs on recombinant jagged-1 increased IntDHS activity and endogenous ARHGAP42 expression while siRNA-mediated knockdown of TEAD1 inhibited ARHGAP42 mRNA levels. Re-chromatin immunoprecipitation experiments indicated that RBPJ and TEAD1 were bound to the Int1DHS enhancer at the same time, and coimmunoprecipitation assays indicated that these factors interacted physically. Our results also suggest TEAD1 and RBPJ bound cooperatively to the Int1DHS and that the presence of TEAD1 promoted the recruitment of NICD by RBPJ. Finally, we showed that ARHGAP42 expression was inhibited by micro-RNA 505 (miR505) which interacted with the ARHGAP42 3'-untranslated region (UTR) to facilitate its degradation and by AK124326, a long noncoding RNA that overlaps with the ARHGAP42 transcription start site on the opposite DNA strand. Since siRNA-mediated depletion of AK124326 was associated with increased H3K9 acetylation and RNA Pol-II binding at the ARHGAP42 gene, it is likely that AK124326 inhibits ARHGAP42 transcription.NEW & NOTEWORTHY First, RBPJ and TEAD1 converge at an intronic enhancer to regulate ARHGAP42 expression in SMCs. Second, TEAD1 and RBPJ interact physically and bind cooperatively to the ARHGAP42 enhancer. Third, miR505 interacts with the ARHGAP42 3'-UTR to facilitate its degradation. Finally, LncRNA, AK124326, inhibits ARHGAP42 transcription.

The Crystalloid Co-Load: Clinically as Effective as Colloid Preload for Preventing Hypotension from Spinal Anaesthesia for Caesarean Delivery.

OBJECTIVE: Colloid preloading diminishes post-spinal hypotension. However, whether colloid preloading is superior to crystalloid co-loading is uncertain. In this retrospective study, we compared the effects of a colloid preload versus a crystalloid co-load on vasopressor requirements and maternal haemodynamics among women undergoing elective caesarean delivery (CD) with spinal anaesthesia. METHODS: We extracted data from the medical records of 160 healthy women who underwent elective CD with spinal anaesthesia at an academic obstetric centre before and after an institutional fluid-loading protocol change. Patients received a 500 mL 6% hydroxyethyl starch preload or a 1000 mL crystalloid co-load. The primary outcome was the total phenylephrine dose administered from spinal block placement to delivery. RESULTS: Our cohort comprised 79 women in the colloid group and 77 women in the crystalloid group. The mean phenylephrine use was significantly lower in the colloid group than in the crystalloid group (489±403 µg vs. 647±464 µg, respectively, p=0.02). The maximal drop in systolic blood pressure was greater in the colloid group than in the crystalloid group (36±20 mmHg vs. 29±16 mmHg, respectively, p=0.02). There were no clinically significant differences between the groups in heart rate, blood loss, temperature and Apgar scores. CONCLUSION: Vasopressor use was lower in colloid preloading than in crystalloid co-loading. However, differences in all outcome measures were minimal and likely clinically insignificant, suggesting that both fluid-loading techniques are appropriate to use for the prevention of spinal hypotension in women undergoing CD.

Druggable targets in the Rho pathway and their promise for therapeutic control of blood pressure.

The prevalence of high blood pressure (also known as hypertension) has steadily increased over the last few decades. Known as a silent killer, hypertension increases the risk for cardiovascular disease and can lead to stroke, heart attack, kidney failure and associated sequela. While numerous hypertensive therapies are currently available, it is estimated that only half of medicated patients exhibit blood pressure control. This signifies the need for a better understanding of the underlying cause of disease and for more effective therapies. While blood pressure homeostasis is very complex and involves the integrated control of multiple body systems, smooth muscle contractility and arterial resistance are important contributors. Strong evidence from pre-clinical animal models and genome-wide association studies indicate that smooth muscle contraction and BP homeostasis are governed by the small GTPase RhoA and its downstream target, Rho kinase. In this review, we summarize the signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity in smooth muscle cells and discuss current therapeutic strategies to target these RhoA pathway components. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations.

A novel familial truncating mutation in the filamin C gene associated with cardiac arrhythmias.

The authors report for the first time a novel mutation in the FLNC gene associated with cardiac arrhythmias in two half-siblings. The FLNC gene on chromosome 7q32 encodes filamin C, which stabilizes the actin network within the cardiomyocyte. The proband is an 8-year-old asymptomatic patient with frequent premature ventricular contractions noted on serial monitoring. Interestingly, the proband and his half-brother harbored a heterozygous 13 base pair deletion that resulted in a frameshift mutation and introduction of a premature stop codon. Notably, the proband also had a very tragic family history of sudden death in young individuals involving three generations and five family members. Because of their concerning family history and arrhythmias, both siblings underwent off-label implantable cardiac device placement for primary prevention of sudden cardiac death. Whether or not the FLNC mutation is associated with sudden cardiac death requires additional investigation and is beyond the scope of this manuscript. While previous studies have identified several mutations in the FLNC gene associated with dilated and hypertrophic cardiomyopathies, the goal of this study was to report a novel mutation in the FLNC gene that is associated with cardiac arrhythmias. The current study indicates that this mutation may help identify patients at risk for cardiac arrhythmias who would benefit from further cardiac evaluation.

Blood pressure-associated polymorphism controls ARHGAP42 expression via serum response factor DNA binding.

We recently demonstrated that selective expression of the Rho GTPase-activating protein ARHGAP42 in smooth muscle cells (SMCs) controls blood pressure by inhibiting RhoA-dependent contractility, providing a mechanism for the blood pressure-associated locus within the ARHGAP42 gene. The goals of the current study were to identify polymorphisms that affect ARHGAP42 expression and to better assess ARHGAP42's role in the development of hypertension. Using DNase I hypersensitivity methods and ENCODE data, we have identified a regulatory element encompassing the ARHGAP42 SNP rs604723 that exhibits strong SMC-selective, allele-specific activity. Importantly, CRISPR/Cas9-mediated deletion of this element in cultured human SMCs markedly reduced endogenous ARHGAP42 expression. DNA binding and transcription assays demonstrated that the minor T allele variation at rs604723 increased the activity of this fragment by promoting serum response transcription factor binding to a cryptic cis-element. ARHGAP42 expression was increased by cell stretch and sphingosine 1-phosphate in a RhoA-dependent manner, and deletion of ARHGAP42 enhanced the progression of hypertension in mice treated with DOCA-salt. Our analysis of a well-characterized cohort of untreated borderline hypertensive patients suggested that ARHGAP42 genotype has important implications in regard to hypertension risk. Taken together, our data add insight into the genetic mechanisms that control blood pressure and provide a potential target for individualized antihypertensive therapies.

Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription.

We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure.

Spinal manipulation for the treatment of hypertension: a systematic qualitative literature review.

OBJECTIVE: Spinal manipulative therapy (SMT) has been reported to successfully treat hypertension (HTN). The purpose of this study was to perform a qualitative literature review on the efficacy of SMT for treating HTN. METHODS: The literature was systematically searched in PubMed, Medline, Allied and Complementary Medicine Database, Cumulative Index to Nursing and Allied Health Literature, and Index of Chiropractic Literature. Included articles were rated for bias using the Cochrane Collaboration's tool for assessing risk of bias. Studies reporting differing methodologies, types of SMT, frequency of treatment, and time of follow-up were considered too dissimilar for meta-analysis. RESULTS: Of 208 articles identified, 10 were selected as relevant and were assessed. Risk of bias scores revealed 2 studies with low risk, 3 studies with unclear risk, and 5 studies with high risk. The maximum improvement observed in any SMT group, in low risk of bias studies was -9.7 (95% confidence interval [CI], -21.1 to 1.8) systolic improvement and -9.0 (95% CI, -16.8 to -1.2) diastolic; and in unclear risk of bias studies, it was -17.2 (95% CI, -20.7 to -13.7) systolic and -13.0 (95% CI, -15.4 to -10.6) diastolic. Statistically significant decreases in blood pressure were not observed in clinical trials with low bias when SMT was compared with effleurage massage and a 5-minute wait. The studies with more risk of bias showed a greater treatment effect. CONCLUSION: There is currently a lack of low bias evidence to support the use of SMT as a therapy for the treatment of HTN. Future investigations may clarify if SMT is effective for treating HTN, either by itself or as an adjunctive therapy, and by which physiologic mechanism this occurs.