Joshua D. Hutcheson

Serotonin receptors and heart valve disease—It was meant 2B

Carcinoid heart disease was one of the first valvular pathologies studied in molecular detail, and early research identified serotonin produced by oncogenic enterochromaffin cells as the likely culprit in causing changes in heart valve tissue. Researchers and physicians in the mid-1960s noted a connection between the use of several ergot-derived medications with structures similar to serotonin and the development of heart valve pathologies similar to those observed in carcinoid patients. The exact serotonergic target that mediated valvular pathogenesis remained a mystery for many years until similar cases were reported in patients using the popular diet drug Fen-Phen in the late 1990s. The Fen-Phen episode sparked renewed interest in serotonin-mediated valve disease, and studies led to the identification of the 5-HT(2B) receptor as the likely molecular target leading to heart valve tissue fibrosis. Subsequent studies have identified numerous other activators of the 5-HT(2B) receptor, and consequently, the use of many of these molecules has been linked to heart valve disease. Herein, we: review the molecular properties of the 5-HT(2B) receptor including factors that differentiate the 5-HT(2B) receptor from other 5-HT receptor subtypes, discuss the studies that led to the identification of the 5-HT(2B) receptor as the mediator of heart valve disease, present current efforts to identify potential valvulopathogens by screening for 5-HT(2B) receptor activity, and speculate on potential therapeutic benefits of 5-HT(2B) receptor targeting.

Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked.

An electrically active microneedle array for electroporation

We have designed and fabricated a microneedle array with electrical functionality with the final goal of electroporating skin’s epidermal cells to increase their transfection by DNA vaccines. The microneedle array was made of polymethylmethacrylate (PMMA) by micromolding technology from a polydimethylsiloxane (PDMS) mold, followed by metal deposition, patterning using laser ablation, and electrodeposition. This microneedle array possessed sufficient mechanical strength to penetrate human skin in vivo and was also able to electroporate both red blood cells and human prostate cancer cells as an in vitro model to demonstrate cell membrane permeabilization. A computational model to predict the effective volume for electroporation with respect to applied voltages was constructed from finite element simulation. This study demonstrates the mechanical and electrical functionalities of the first MEMS-fabricated microneedle array for electroporation, designed for DNA vaccine delivery.

Small entities with large impact: microcalcifications and atherosclerotic plaque vulnerability.

Purpose of review Atherosclerotic plaque rupture and subsequent acute events, such as myocardial infarction and stroke, contribute to the majority of cardiovascular-related deaths. Calcification has emerged as a significant predictor of cardiovascular morbidity and mortality, challenging previously held notions that calcifications stabilize atherosclerotic plaques. In this review, we address this discrepancy through recent findings that not all calcifications are equivalent in determining plaque stability. Recent findings The risk associated with calcification is inversely associated with calcification density. As opposed to large calcifications that potentially stabilize the plaque, biomechanical modeling indicates that small microcalcifications within the plaque fibrous cap can lead to sufficient stress accumulation to cause plaque rupture. Microcalcifications appear to derive from matrix vesicles enriched in calcium-binding proteins that are released by cells within the plaque. Clinical detection of microcalcifications has been hampered by the lack of imaging resolution required for in-vivo visualization; however, recent studies have demonstrated promising new techniques to predict the presence of microcalcifications. Summary Microcalcifications play a major role in destabilizing atherosclerotic plaques. The identification of critical characteristics that lead to instability along with new imaging modalities to detect their presence in vivo may allow early identification and prevention of acute cardiovascular events.

Changes in cell morphology due to plasma membrane wounding by acoustic cavitation.

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphologic changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced nonlytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure.

Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

Vascular calcification is a common feature of major cardiovascular diseases. Extracellular vesicles participate in the formation of microcalcifications that are implicated in atherosclerotic plaque rupture; however, the mechanisms that regulate formation of calcifying extracellular vesicles remain obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin regulated the loading of the calcification protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring its calcification potential. Furthermore, SMC calcification required Rab11-dependent trafficking and FAM20C/casein kinase 2-dependent C-terminal phosphorylation of sortilin. In a murine model, Sort1-deficiency reduced arterial calcification but did not affect bone mineralization. Additionally, transfer of sortilin-deficient BM cells to irradiated atherosclerotic mice did not affect vascular calcification, indicating a primary role of SMC-derived sortilin. Together, the results of this study identify sortilin phosphorylation as a potential therapeutic target for ectopic calcification/microcalcification and may clarify the mechanism that underlies the genetic association between the SORT1 gene locus and coronary artery calcification.

5-HT2B antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT(2B)) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT(2B) opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT(2B) antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT(2B) antagonism. Interestingly, 5-HT(2B) antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT(2B) antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT(2B) antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT(2B) antagonism as a novel therapeutic approach for CAVD that merits further investigation.

Cadherin-11 Regulates Cell–Cell Tension Necessary for Calcific Nodule Formation by Valvular Myofibroblasts

Objective—Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MAPK Erk kinase (MEK)1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of transforming growth factor &bgr;1–induced cadherin-11 expression in calcific nodule formation. Methods and Results—As shown previously, porcine AVICs treated with transforming growth factor &bgr;1 before cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, transforming growth factor &bgr;1–treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in transforming growth factor &bgr;1–treated AVICs possessing cadherin-11. Furthermore, when small interfering RNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell–cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets. Conclusion—These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2-dependent pathway.

MicroRNA in cardiovascular calcification: focus on targets and extracellular vesicle delivery mechanisms.

Cardiovascular calcification is a prominent feature of chronic inflammatory disorders—such as chronic kidney disease, type 2 diabetes mellitus, and atherosclerosis—that associate with significant morbidity and mortality. The concept that similar pathways control both bone remodeling and vascular calcification is widely accepted, but the precise mechanisms of calcification remain largely unknown. The central role of microRNAs (miRNA) as fine-tune regulators in the cardiovascular system and bone biology has gained acceptance and has raised the possibility for novel therapeutic targets. Additionally, circulating miRNAs have been proposed as biomarkers for a wide range of cardiovascular diseases, but knowledge of miRNA biology in cardiovascular calcification is very limited. This review focuses on the role of miRNAs in cardiovascular disease, with emphasis on osteogenic processes. Herein, we discuss the current understanding of miRNAs in cardiovascular calcification. Furthermore, we identify a set of miRNAs common to diseases associated with cardiovascular calcification (chronic kidney disease, type 2 diabetes mellitus, and atherosclerosis), and we hypothesize that these miRNAs may provide a molecular signature for calcification. Finally, we discuss this novel hypothesis with emphasis on known biological and pathological osteogenic processes (eg, osteogenic differentiation, release of calcifying matrix vesicles). The aim of this review is to provide an organized discussion of the known links between miRNA and calcification that provide emerging concepts for future studies on miRNA biology in cardiovascular calcification, which will be critical for developing new therapeutic strategies.

SAVING CELLS FROM ULTRASOUND-INDUCED APOPTOSIS: QUANTIFICATION OF CELL DEATH AND UPTAKE FOLLOWING SONICATION AND EFFECTS OF TARGETED CALCIUM CHELATION

Applications of ultrasound for noninvasive drug and gene delivery have been limited by associated cell death as a result of sonication. In this study, we sought to quantify the distribution of cellular bioeffects caused by low-frequency ultrasound (24 kHz) and test the hypothesis that Ca(2+) chelation after sonication can shift this distribution by saving cells from death by apoptosis. Using flow cytometry, we quantitatively categorized sonicated cells among four populations: (i) cells that appear largely unaffected, (ii) cells reversibly permeabilized, (iii) cells rendered nonviable during sonication and (iv) cells that appear to be viable shortly after sonication, but later undergo apoptosis and die. By monitoring cells for 6 h after ultrasound exposure, we found that up to 15% of intact cells fell into this final category. Those apoptotic cells initially had the highest levels of uptake of a marker compound, calcein; also had highly elevated levels of intracellular Ca(2+); and contained an estimated plasma membrane wound radius of 100-300 nm. Finally, we showed that chelation of intracellular Ca(2+) after sonication reduced apoptosis by up to 44%, thereby providing a strategy to save cells. We conclude that cells can be saved from ultrasound-induced death by appropriate selection of ultrasound conditions and Ca(2+) chelation after sonication.