Adam W. Akerman

HDACs Regulate miR-133a Expression in Pressure Overload–Induced Cardiac Fibrosis

Background—MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. Methods and Results—Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload hypertrophy. Mice were treated with class I and IIb HDAC inhibitor (HDACi) via drinking water for 2 and 4 weeks post TAC. miRNA expression was determined by real-time polymerase chain reaction. Echocardiography was performed at baseline and post TAC end points for structural and functional assessment. Chromatin immunoprecipitation was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7- and 0.5-fold at 2 and 4 weeks post TAC, respectively, when compared with vehicle control (P<0.05). HDAC inhibition prevented this significant decrease 2 weeks post TAC and maintained miR-133a expression near vehicle control levels, which coincided with (1) a decrease in connective tissue growth factor expression, (2) a reduction in cardiac fibrosis and left atrium diameter (marker of end-diastolic pressure), suggesting an improvement in diastolic function. Chromatin immunoprecipitation analysis revealed that HDAC1 and HDAC2 are present on the miR-133a enhancer regions. Conclusions—The results reveal that HDACs play a role in the regulation of pressure overload–induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload–induced cardiac fibrosis.

MicroRNAs emerging as mediators of remodeling with atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is now established as an independent risk factor for stroke. Moreover, a concomitant diagnosis of AF greatly complicates treatment for a number of disease processes such as diabetes and congestive heart failure. Given the recognized additional burden that AF places on the health-care system, significant research has been performed in an attempt to delineate mechanisms that contribute to AF initiation as well as progression. Understandably, there is an extensive body of research that has identified abnormalities in ionic channels/electrogenic processes that occur with AF (reviewed in References 2 and 3). For example, abnormalities in the abundance of sodium, potassium, and calcium channels have been reported with AF. Therefore, the determination of cellular mechanisms that regulate protein abundance may shed new light on the pathogenesis of AF. Recent interest in the field of regulation of protein abundance has focused on microRNAs, which are small, single-stranded, noncoding regulatory RNAs approximately 19–23 nucleotides in length. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. MicroRNAs are transcribed in the nucleus and then undergo a multistepped “maturation” process involving truncation, exportation from the nucleus, and incorporation into a complex (termed the RNA-induced silencing complex [RISC]) that includes endonuclease activity from the Argonaute family of proteins. Once incorporated into a RISC, the microRNAs regulate the targeted mRNA by degradation either through direct cleavage or by inhibiting protein synthesis. Therefore, there is—almost always—a negative relationship between the levels of a particular microRNA and the abundance of the protein(s) targeted by that microRNA. The impact of microRNA-mediated regulation of cellular/ extracellular processes is only just being realized. Presently, over 2000 human microRNA sequences have been identified (miRBase, Release 19), and these numbers are steadily

Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms

Objective: Membrane type‐1 matrix metalloproteinase (MT1‐MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)‐2 and the release of matrix‐ bound transforming growth factor‐&bgr;. In this study, we tested the hypothesis that MT1‐MMP is subject to protein kinase C (PKC)–mediated regulation, which alters intracellular trafficking and activity with TAAs. Methods: Levels of MMP‐2, native and phosphorylated MT1‐MMP, and PKC‐&dgr; were measured in aortic tissue from patients with small TAAs (<5 cm; n = 8) and large TAAs (>6.5 cm; n = 8), and compared with values measured in normal controls (n = 8). Cellular localization of green fluorescent protein (GFP)‐tagged MT1‐MMP was assessed in aortic fibroblasts isolated from control and 4‐week TAA mice. The effects of PKC‐mediated phosphorylation on MT1‐MMP cellular localization and function (active MMP‐2 vs phospo‐Smad2 abundance) were assessed after treatment with a PKC activator (phorbol‐12‐myristate‐13‐acetate [PMA], 100 nM) with and without a PKC‐&dgr;–specific inhibitor (röttlerin, 3 &mgr;M). Results: Compared with controls, MT1‐MMP abundance was increased in aortas from both TAA groups. Active MMP‐2 was increased only in the large TAA group. The abundances of phosphorylated MT1‐MMP and activated PKC‐&dgr; were enhanced in the small TAA group compared with the large TAA group. MT1‐MMP was localized on the plasma membrane in aortic fibroblasts from control mice and in endosomes from TAA mice. Treatment with PMA induced MT1‐MMP‐GFP internalization, enhanced phospho‐Smad2, and reduced MMP‐2 activation, whereas röttlerin pretreatment inhibited these effects. Conclusions: Phosphorylation of MT1‐MMP mediates its activity through directing cellular localization, shifting its role from MMP‐2 activation to intracellular signaling. Thus, targeted inhibition of MT1‐MMP may have therapeutic relevance as an approach to attenuating TAA development.

Differential hypertensive protease expression in the thoracic versus abdominal aorta

Background Hypertension (HTN), which is a major risk factor for cardiovascular morbidity and mortality, can drive pathologic remodeling of the macro‐ and microcirculation. Patterns of aortic pathology differ, however, suggesting regional heterogeneity of the pressure‐sensitive protease systems triggering extracellular matrix remodeling in the thoracic (TA) and abdominal aortas (AA). This study tested the hypothesis that the expression of two major protease systems (matrix metalloproteinases [MMPs] and cathepsins) in the TA and AA would be differentially affected with HTN. Methods Normotensive (BPN3) mice at 14‐16 weeks of age underwent implantation of osmotic infusion pumps for 28‐day angiotensin II (AngII) delivery (1.46 mg/kg/day; BPN3+AngII; n = 8) to induce HTN. The TA and AA were harvested to determine levels of MMP‐2, MMP‐9, and membrane type 1‐MMP, and cathepsins S, K, and L were evaluated in age‐matched BPN3 (n = 8) control and BPH2 spontaneously hypertensive mice (non‐AngII pathway; n = 7). Blood pressure was monitored via CODA tail cuff plethysmography (Kent Scientific Corporation, Torrington, Conn). Quantitative real‐time polymerase chain reaction and immunoblotting/zymography were used to measure MMP and cathepsin messenger RNA expression and protein abundance, respectively. Target protease values were compared within each aortic region via analysis of variance. Results Following 28 days infusion, the BPN3+AngII mice had a 17% increase in systolic blood pressure, matching that of the BPH2 spontaneously hypertensive mice (both P < .05 vs BPN3). MMP‐2 gene expression demonstrated an AngII‐dependent increase in the TA (P < .05), but MMP‐9 was not altered with HTN. Expression of tissue inhibitor of metalloproteinases‐1 was markedly increased in TA of BPN3+AngII mice, but tissue inhibitor of metalloproteinases‐2 demonstrated decreased expression in the AA of both hypertensive groups (P < .05). Only cathepsin K responded to AngII‐induced HTN with significant elevation in the TA of those mice, but expression of cathepsin L and cystatin C was inhibited in AA of both hypertensive groups (P < .05). Apoptotic markers were not significantly elevated in any experimental group. Conclusions By using two different models of HTN, this study has identified pressure‐dependent as well as AngII‐dependent regional alterations in aortic gene expression of MMPs and cathepsins that may lead to differential remodeling responses in each of the aortic regions. Further studies will delineate mechanisms and may provide targeted therapies to attenuate down‐stream aortic pathology based on demonstrated regional heterogeneity. Clinical Relevance Hypertension represents a primary risk factor for cardiovascular morbidity and mortality. Given the epidemiologic association with aortic aneurysms, interest has been generated regarding whether the hypertensive state creates an environment in the aortic media that is vulnerable to degenerative remodeling. This investigation has been initiated by exploring two major protease systems, the matrix metalloproteinases and the cathepsins, and addresses the hypothesis that hypertension differentially regulates the expression of two major protease systems in the thoracic aorta vs abdominal aorta. Understanding region‐specific protease expression may allow for engineering of targeted aortic therapy.