Edie C. Goldsmith

Organization of fibroblasts in the heart.

Cardiac fibroblasts are organized into a three‐dimensional network in the heart. This organization follows the endomysial weave network that surrounds groups of myocytes. Reverse transcriptase‐polymerase chain reaction, Western blots, and immunohistochemistry were used to show that discoidin domain receptor 2 (DDR2) was specific for cardiac fibroblasts and not expressed on endothelial cells, smooth muscle cells, or cardiac myocytes. DDR2 is expressed early in development and in the adult heart. High voltage electron microscopy (HVEM), scanning electron microscopy, and laser scanning confocal microscopy document the three‐dimensional organization of fibroblasts in the heart. Antibodies against connexin 43 and 45 showed different patterns but confirmed, along with HVEM, that fibroblasts are connected to each other as well as cardiac myocytes. The implications of this arrangement of fibroblasts can be important to cardiac function. The signaling of DDR2 and the expression of matrix metalloproteinase 2 in relation to collagen turnover and remodeling is discussed. Developmental Dynamics 230:787–794, 2004.

Left Ventricular Hypertrophy in Ascending Aortic Stenosis Mice Anoikis and the Progression to Early Failure

BACKGROUND To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice. METHODS AND RESULTS Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7+/-0.7 versus 3.1+/-0.2 and 5. 7+/-0.4 versus 2.7+/-0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0+/-0.8 and 25.2+/-1.8 versus 14. 1+/-0.5 microm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for beta(1)-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides. CONCLUSIONS Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.

Specialization at the Z line of cardiac myocytes

Time for primary review 28 days. The organization of any differentiated cell is not random but is the result of a dynamic integration of extracellular and intracellular signals. During the development of the heart, cardiac myocytes are round shaped cells that differentiate into a rod-shaped phenotype. During the different stages of commitment and morphogenesis, the myocyte organizes its internal structure by undergoing myofibrillogenesis. This process results in the precise arrangement of contractile elements, supporting cytoskeleton, and endoplasmic reticulum. Within the sarcolemma, specialized regions are defined for attachment to components of the extracellular matrix (ECM). This specialized site of the sarcolemma consists of the ECM—Receptor—Cytoskeleton complex (Fig. 1). These sites will integrate attachment to the ECM and the series of proteins necessary for the chemical and mechanical transmission of information. In this review, we will describe the evidence that indicates there is a specialization of the sarcolemma at the Z line for the clustering of various receptors for the ECM as well as the cytoskeleton. Fig. 1 A diagrammatic representation of the specialized regions of the sarcolemma. For cell—cell attachment, the extracellular regions of cadherin molecules exhibit homotypic binding while the cytoplasmic domains connect to catenins and actin. A variety of receptors including integrins and growth factors are found in a specialized region on the lateral margin of the myocyte at or near the Z band. Both complexes have direct associations with various signal transduction and cytoskeletal components. It appears that the lateral complexes are not associated with actin like the cadherin—catenin complex. During development of the embryonic heart, two specialized regions of the sarcolemma develop: (1) intercalated disks for cell—cell interaction and (2) ECM—integrin—cytoskeletal connections for cell—ECM contacts. The formation of these regions appears to be a coordinated expression of ECM components, cell surface receptors, and signaling proteins [1–5]. The localization … * Corresponding author. Tel.: +1-803-733-3115; fax: +1-803-733-1533 borg{at}med.sc.edu

Polyelectrolyte-coated gold nanorods and their interactions with type I collagen

Gold nanorods (AuNRs) have unique optical properties for numerous biomedical applications, but the interactions between AuNRs and proteins, particularly those of the extracellular matrix (ECM), are poorly understood. Here the effects of AuNRs on the self-assembly, mechanics, and remodeling of type I collagen gels were examined in vitro. AuNRs were modified with polyelectrolyte multilayers (PEMs) to minimize cytotoxicity, and AuNRs with different terminal polymer chemistries were examined for their interactions with collagen by turbidity assays, rheological tests, and microscopy. Gel contraction assays were used to examine the effects of the PEM-coated AuNRs on cell-mediated collagen remodeling. Polyanion-terminated AuNRs significantly reduced the lag (nucleation) phase of collagen self-assembly and significantly increased the dynamic shear modulus of the polymerized gels, whereas polycation-terminated AuNRs had no effect on the mechanical properties of the collagen. Both polyanion- and polycation-terminated AuNRs significantly inhibited collagen gel contraction by cardiac fibroblasts, and the nanoparticles were localized in intra-, peri-, and extracellular compartments, suggesting that PEM-coated AuNRs influence cell behavior via multiple mechanisms. These results demonstrate the significance of nanoparticle-ECM interactions in determining the bioactivity of nanoparticles.

Expression of Discoidin Domain Receptor 2 (DDR2) in the Developing Heart

Interactions between cells and the surrounding extracellular matrix are important for a number of developmental events. In the heart, cardiac fibroblasts produce the majority of extracellular matrix proteins, particularly collagen types I and III. Cells originating from the proepicardial organ migrate over the surface of the heart, invade the underlying myocardium and ultimately give rise to smooth muscle cells, fibroblasts, and coronary endothelium. Although integrin expression in the developing heart has been well characterized, the expression of Discoidin Domain Receptor 2 (DDR2) remains to be defined. Using confocal microscopy, the expression of DDR2 was examined at several points during cardiac development. Initially, DDR2 expression was detected on the epicardial surface of the heart and on endothelial and mesenchymal cells within the cardiac cushions. As development progressed, DDR2 expression increased at localized regions in the apex and atrioventricular sulcus, although this expression decreased from epicardial to endocardial surface. Eventually, DDR2 expression spanned the myocardial free wall and was detected within the septum. Not until postnatal development was DDR2 expression detected uniformly throughout the myocardium and this distribution was maintained in the adult heart. In summary, the data presented demonstrate that the distribution of DDR2-positive cells changes within the heart during development.

The effect of gold nanorods on cell-mediated collagen remodeling

Cardiac fibroblasts, the noncontractile cells of the heart, contribute to myocardial maintenance through the deposition, degradation, and organization of collagen. Adding polyelectrolyte-coated gold nanorods to three-dimensional constructs composed of collagen and cardiac fibroblasts reduced contraction and altered the expression of mRNAs encoding beta-actin, alpha-smooth muscle actin, and collagen type I. These data show that nanomaterials can modulate cell-mediated matrix remodeling and suggest that the targeted delivery of nanomaterials can be applied for antifibrotic therapies.

Regulation of tissue fibrosis by the biomechanical environment.

The biomechanical environment plays a fundamental role in embryonic development, tissue maintenance, and pathogenesis. Mechanical forces play particularly important roles in the regulation of connective tissues including not only bone and cartilage but also the interstitial tissues of most organs. In vivo studies have correlated changes in mechanical load to modulation of the extracellular matrix and have indicated that increased mechanical force contributes to the enhanced expression and deposition of extracellular matrix components or fibrosis. Pathological fibrosis contributes to dysfunction of many organ systems. A variety of in vitro models have been utilized to evaluate the effects of mechanical force on extracellular matrix-producing cells. In general, application of mechanical stretch, fluid flow, and compression results in increased expression of extracellular matrix components. More recent studies have indicated that tissue rigidity also provides profibrotic signals to cells. The mechanisms whereby cells detect mechanical signals and transduce them into biochemical responses have received considerable attention. Cell surface receptors for extracellular matrix components and intracellular signaling pathways are instrumental in the mechanotransduction process. Understanding how mechanical signals are transmitted from the microenvironment will identify novel therapeutic targets for fibrosis and other pathological conditions.

Light scattering from gold nanorods: tracking material deformation

We demonstrate the use of optical patterns, produced by resonant Rayleigh scattering from gold nanorods, as markers by which local deformations can be measured using image correlation techniques. While the use of optical data, in this case from dark-field microscopy, to generate deformational field information (displacements and strains) is not new, the use of the light scattered from gold nanorods as the correlated pattern is new, and has the potential to enable smaller scale measurements even over large deformations. We find excellent agreement between the measured and theoretical deformation and strain fields for two sample polymers with gold nanorod markers. The gold nanorod surface can be modified to make biocompatible nanomaterials, which will be useful for examining mechanical effects in biological tissue.

Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts

AIMS Cardiovascular disease is the leading cause of death for individuals diagnosed with type II diabetes mellitus (DM). Changes in cardiac function, left ventricular wall thickness and fibrosis have all been described in patients and animal models of diabetes; however, the factors mediating increased matrix deposition remain unclear. The goal of this study was to evaluate whether cardiac fibroblast function is altered in a rat model of type II DM. MAIN METHODS Cardiac fibroblasts were isolated from 14 week old Zucker diabetic and lean control (LC) adult male rat hearts. Fibroblasts were examined for their ability to remodel 3-dimensional collagen matrices, their adhesion, migration and proliferation on collagen and changes in gene expression associated with collagen remodeling. KEY FINDINGS Cardiac fibroblasts from diabetic animals demonstrated significantly greater ability to contract 3-dimensional collagen matrices compared to cardiac fibroblasts from LC animals. The enhanced contractile behavior was associated with an increase in diabetic fibroblast proliferation and elevated expression of α-smooth muscle actin and type I collagen, suggesting the transformation of diabetic fibroblasts into a myofibroblast phenotype. SIGNIFICANCE Cardiac fibrosis is a common complication in diabetic cardiomyopathy which may contribute to the observed cardiac dysfunction associated with this disease. Identifying and understanding the changes in fibroblast behavior which contribute to the increased deposition of collagen and other matrix proteins may provide novel therapeutic targets for reducing the devastating effects of diabetes on the heart.