Patrick Collier

Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype

Ischemia caused by coronary artery disease and myocardial infarction leads to aberrant ventricular remodeling and cardiac fibrosis. This occurs partly through accumulation of gene expression changes in resident fibroblasts, resulting in an overactive fibrotic phenotype. Long-term adaptation to a hypoxic insult is likely to require significant modification of chromatin structure in order to maintain the fibrotic phenotype. Epigenetic changes may play an important role in modulating hypoxia-induced fibrosis within the heart. Therefore, the aim of the study was to investigate the potential pro-fibrotic impact of hypoxia on cardiac fibroblasts and determine whether alterations in DNA methylation could play a role in this process. This study found that within human cardiac tissue, the degree of hypoxia was associated with increased expression of collagen 1 and alpha-smooth muscle actin (ASMA). In addition, human cardiac fibroblast cells exposed to prolonged 1% hypoxia resulted in a pro-fibrotic state. These hypoxia-induced pro-fibrotic changes were associated with global DNA hypermethylation and increased expression of the DNA methyltransferase (DNMT) enzymes DNMT1 and DNMT3B. Expression of these methylating enzymes was shown to be regulated by hypoxia-inducible factor (HIF)-1α. Using siRNA to block DNMT3B expression significantly reduced collagen 1 and ASMA expression. In addition, application of the DNMT inhibitor 5-aza-2-deoxycytidine suppressed the pro-fibrotic effects of TGFβ. Epigenetic modifications and changes in the epigenetic machinery identified in cardiac fibroblasts during prolonged hypoxia may contribute to the pro-fibrotic nature of the ischemic milieu. Targeting up-regulated expression of DNMTs in ischemic heart disease may prove to be a valuable therapeutic approach.

Can emerging biomarkers of myocardial remodelling identify asymptomatic hypertensive patients at risk for diastolic dysfunction and diastolic heart failure

Hypertension is one of the main drivers of the heart failure (HF) epidemic. The aims of this study were to profile fibro‐inflammatory biomarkers across stages of the hypertensive heart disease (HHD) spectrum and to examine whether particular biochemical profiles in asymptomatic patients identify a higher risk of evolution to HF.

Proteomic Analysis of Coronary Sinus Serum Reveals Leucine-Rich α2-Glycoprotein as a Novel Biomarker of Ventricular Dysfunction and Heart Failure

Background—Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF. Methods and Results—Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich &agr;2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P⩽0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-&agr; (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-&bgr;R1 (P<0.001) and &agr;-smooth muscle actin (P=0.025) expression. Conclusions—LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, &bgr;-blocker therapy, and BNP.

A Test in Context: Myocardial Strain Measured by Speckle-Tracking Echocardiography

Strain-based imaging techniques (and specifically speckle-tracking echocardiography) have been shown to have clinical utility in a variety of settings. This technique is being embraced and increasingly adopted in many echocardiography laboratories worldwide. This review appraised speckle-tracking echocardiography in a clinical context by providing a critical evaluation of the prognostic and diagnostic insights that this technology can provide. In particular, we discuss the use of speckle-tracking strain in selected areas, such as undifferentiated left ventricular hypertrophy, cardio-oncology, aortic stenosis, and ischemic heart disease. The potential utility of regional and chamber strains (namely segmental left ventricular strain, left atrial strain, and right ventricular strain) are also discussed. Future directions for this technology are explored. Before its clinical application, it is particularly important that physicians be cognizant of the technical challenges and inherent limitations of strain data, which are also addressed here.

Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts: a possible defense against transforming growth factor-β mediated fibrosis

BackgroundMechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts’ response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.Methods and resultsThe effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.ConclusionWe postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Proteomic Analysis of Coronary Sinus Serum Reveals LRG as a Novel Biomarker of Ventricular Dysfunction and Heart Failure

Background—Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF. Methods and Results—Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich &agr;2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P⩽0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-&agr; (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-&bgr;R1 (P<0.001) and &agr;-smooth muscle actin (P=0.025) expression. Conclusions—LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, &bgr;-blocker therapy, and BNP.

Modest elevation in BNP in asymptomatic hypertensive patients reflects sub-clinical cardiac remodeling, inflammation and extracellular matrix changes.

In asymptomatic subjects B-type natriuretic peptide (BNP) is associated with adverse cardiovascular outcomes even at levels well below contemporary thresholds used for the diagnosis of heart failure. The mechanisms behind these observations are unclear. We examined the hypothesis that in an asymptomatic hypertensive population BNP would be associated with sub-clinical evidence of cardiac remodeling, inflammation and extracellular matrix (ECM) alterations. We performed transthoracic echocardiography and sampled coronary sinus (CS) and peripheral serum from patients with low (nu200a=u200a14) and high BNP (nu200a=u200a27). Peripheral BNP was closely associated with CS levels (ru200a=u200a0.92, p<0.001). CS BNP correlated significantly with CS levels of markers of collagen type I and III turnover including: PINP (ru200a=u200a0.44, pu200a=u200a0.008), CITP (ru200a=u200a0.35, pu200a=u200a0.03) and PIIINP (ru200a=u200a0.35, pu200a=u200a0.001), and with CS levels of inflammatory cytokines including: TNF-α (ru200a=u200a0.49, pu200a=u200a0.002), IL-6 (ru200a=u200a0.35, pu200a=u200a0.04), and IL-8 (ru200a=u200a0.54, p<0.001). The high BNP group had greater CS expression of fibro-inflammatory biomarkers including: CITP (3.8±0.7 versus 5.1±1.9, pu200a=u200a0.007), TNF-α (3.2±0.5 versus 3.7±1.1, pu200a=u200a003), IL-6 (1.9±1.3 versus 3.4±2.7, pu200a=u200a0.02) and hsCRP (1.2±1.1 versus 2.4±1.1, pu200a=u200a0.04), and greater left ventricular mass index (97±20 versus 118±26 g/m2, pu200a=u200a0.03) and left atrial volume index (18±2 versus 21±4, pu200a=u200a0.008). Our data provide insight into the mechanisms behind the observed negative prognostic impact of modest elevations in BNP and suggest that in an asymptomatic hypertensive cohort a peripheral BNP measurement may be a useful marker of an early, sub-clinical pathological process characterized by cardiac remodeling, inflammation and ECM alterations.

Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50xa0%, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70xa0%). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (nu2009=u200910) and HF (nu2009=u20096) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Abstract Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.