PubMed Health⌕ Search

Biomedical subjects

T K Borg

Publications and source records attributed to T K Borg.

At least 19 recordsLinked to original sources
PubMed2002

Isotonic biaxial loading of fibroblast-populated collagen gels: a versatile, low-cost system for the study of mechanobiology.

We developed a simple, versatile system for applying a range of biaxial loads to cell-matrix constructs for the study of mechanobiology. The system consists of porous polyethylene bars that are polymerized into a square fibroblast-populated gel and loaded by freely hanging weights attached to sutures routed through a custom loading rig. The cost to manufacture each mold/loading rig pair was less than US dollars 250 and the expected life of the components is up to 10 years. Neonatal and adult cardiac fibroblasts contracted gels to a decreasing extent as external load was increased ( P=0.003) and achieved contraction forces of up to 1.4 mN per million cells. Strain distributions were reasonably homogeneous in the central region of the gel (25% of gel area), but clearly nonhomogeneous outside that central region. The primary advantages of this system are simplicity, low cost, biaxial loading, and the ability to test for a dose-response effect of mechanical load. The current disadvantages are the inability to apply cycling loading and the inhomogeneities introduced by the use of rigid loading bars.

Animals↗
PubMed2001

Integrins and the myocardium.

Extracellular matrix provides a structural, chemical, and mechanical substrate that is essential in cardiac development, growth, and responses to pathophysiological signals. Transmembrane receptors termed integrins provide a dynamic interaction of environmental cues and intracellular events. Integrins orchestrate multiple functions in the intact organism including organogenesis, regulation of gene expression, cell proliferation, differentiation, migration, and death. They are expressed in all cellular components of the cardiovascular system, including the vasculature, blood, cardiac myocytes and nonmuscle cardiac cells. The focus of this review will be on the role of integrins in the myocardium. We will provide background on integrin structure and function, discuss how the expression of integrins is critical to the form and function of the developing and postnatal myocardium, and review the known data on integrins as signaling molecules in the heart. Finally, we will offer insights to the future research directions into this important family of extracellular matrix receptors in the myocardium.

Animals↗
PubMed2001

The collagenous domain of class A scavenger receptors is involved in macrophage adhesion to collagens.

Class A macrophage scavenger receptors (MSRs) have a remarkably broad ligand specificity and are well-known for their roles in atherogenesis and host defense. Recently, we demonstrated that these receptors also recognize and mediate adhesion to denatured forms of type I collagen. In this study, the involvement of the collagenous domain of MSRs in binding to denatured type I collagen was investigated. Transient expression of full-length, native type II MSR in COS-1 cells conferred adhesion to denatured type I collagens, whereas expression of a truncated receptor lacking the distal portion of the collagenous domain did not. Further, a synthetic peptide derived from the collagenous domain was effective in abrogating Mphi adhesion to denatured forms of type I collagen. We also addressed collagen-type specificity by examining MSR affinity for type III and type IV collagens. As with type I collagen, Mphis adhered only to denatured forms of type III collagen. Moreover, the adhesion was mediated by MSRs. In contrast, adhesion to denatured type IV collagen was not shown to be MSR-dependent, but adhesion to the native form was. MSR-mediated adhesion to types III and IV collagens was also shown to be dependent on the collagenous domain. Taken together, these data strongly suggest that the collagenous domain is involved in MSR-mediated adhesion to denatured forms of types I and III collagens and native, but not denatured, type IV collagen.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
PubMed2000

Selective adhesion of macrophages to denatured forms of type I collagen is mediated by scavenger receptors.

Macrophages (M&phi;s) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, M&phi;s reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by M&phi;s is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors.

Animals↗
PubMed2000

Design and construction of a uniaxial cell stretcher.

In vitro mechanical cell stimulators are used for the study of the effect of mechanical stimulation on anchorage-dependent cells. We developed a new mechanical cell stimulator, which uses stepper motor technology and computer control to achieve a high degree of accuracy and repeatability. This device also uses high-performance plastic components that have been shown to be noncytotoxic, dimensionally stable, and resistant to chemical degradation from common culture laboratory chemicals. We show that treatment with glow discharge for 25 s at 20 mA is sufficient to modify the surface of the rubber to allow proper adhesion for polymerization of aligned collagen. We show through finite element analysis that the middle area of the membrane, away from the clamped ends, is predictable, homogeneous, and has negligible shear strain. To test the efficacy of the mechanical stretch, we examined the effect of mechanical stimulation on the production of beta(1)-integrin by neonatal rat cardiac fibroblasts. Mechanical stimulation was tested in the range of 0-12% stretch and 0-10-cycles/min stretch frequency. The fibroblasts respond with an increase in beta(1)-integrin at 3% stretch and a decrease at 6 and 12% stretch. Stretch frequency was found to not significantly effect the concentration of beta(1)-integrin. These studies yield a new and improved mechanical cell stimulator and demonstrate that mechanical stimulation has an effect on the expression of beta(1)-integrin.

Animals↗
PubMed2000

Modulation of heart fibroblast migration and collagen gel contraction by IGF-I.

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor-beta, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.

Animals↗
PubMed1999

Regulation of cardiac myocyte protein turnover and myofibrillar structure in vitro by specific directions of stretch.

We have examined how different degrees (0.5%, 1.0%, 2.5%, 5.0%, and 10.0%) and directions of stretch regulate the turnover and accumulation of contractile proteins in cultured neonatal cardiac myocytes (NCMs). In pulse-chase experiments, stellate-shaped NCMs with random arrays of myofibrils (MFs) exhibited a threshold response to stretch. With respect to unstretched controls, the turnover of the contractile protein pool was suppressed 50% to 100% in stellate NCMs stretched 1.0% to 5.0% and was unaltered in stellate NCMs stretched 0.5% or 10.0%. The posttranslational metabolism of myosin heavy chain (MHC) and actin was regulated in parallel with the total contractile protein pool. The turnover of the cytoplasmic protein pool remained unchanged in response to stretch. NCMs plated onto an aligned matrix of type I collagen expressed an elongated, rod-like cell shape. The MFs of these cells were distributed in parallel with one another along a single unique axis. The tissue-like pattern of organization of these cultures made it possible to assay how specific directions of stretch affected cardiac protein turnover and MF organization. In pulse-chase experiments, stretch in parallel with the MFs did not alter the turnover of the total contractile protein pool, the cytoplasmic protein pool, MHC, or actin. The total cellular concentration of MHC and actin remained constant, and MF alignment was not overtly affected. In contrast, even modest degrees of stretch across the short axis of the MFs suppressed total contractile protein turnover, the turnover of MHC and actin, and promoted the accumulation of these MF subunits. The parallel alignment of MFs deteriorated in myocytes stretched greater than 5%. The characteristic response of aligned myocytes to stretch was not affected by the contractile state of the cells. Isoproterenol (ISO) treatment in concert with stretch in parallel with the MFs modestly accelerated contractile protein turnover. Conversely, contractile protein turnover was suppressed in cells treated with ISO and stretched across the short axis of the MFs. Contractile arrest with nifedipine (NIFED) accelerated total myofibrillar protein turnover. Stretch across the short axis, but not in parallel with the MFs, suppressed protein turnover in cells treated with NIFED. The turnover of the cytosolic proteins remained constant under all conditions assayed. These data suggest that specific directions of stretch may play a crucial role in regulating MF organization and the metabolism of contractile proteins in the cardiac myocyte.

Actins↗
PubMed1999

Pressure overload induces severe hypertrophy in mice treated with cyclosporine, an inhibitor of calcineurin.

Cardiac hypertrophy is the fundamental adaptation of the adult heart to mechanical load. Recent work has shown that inhibition of calcineurin activity with cyclosporine suppresses the development of hypertrophy in calcineurin transgenic mice and in in vitro systems of neonatal rat cardiocytes stimulated with peptide growth factors. To test the hypothesis that the calcineurin signaling pathway is critical for load-induced hypertrophy in vivo, we examined the effects of cyclosporine treatment on left ventricular hypertrophy induced by experimental ascending aortic stenosis for 4 weeks in mice. Left ventricular systolic pressure was elevated to a similar level in aortic stenosis mice that were treated with cyclosporine versus no drug. Left ventricular mass and myocyte size were similar in treated and untreated aortic stenosis animals and significantly greater than control animals, showing that cyclosporine treatment does not suppress hypertrophic growth. Both treated and untreated animals showed increased left ventricular expression of the load-sensitive gene atrial natriuretic factor. Calcineurin activity was measured in the left ventricle and the spleen from control mice and aortic stenosis mice treated with cyclosporine versus no drug. Levels of calcineurin activity were similar in the spleens of control and untreated aortic stenosis mice. However, calcineurin activity was severely depressed in left ventricular tissue of untreated aortic stenosis mice compared with control mice and was further reduced by cyclosporine treatment. Thus, pathological hypertrophy and cardiac-restricted gene expression induced by pressure overload in vivo are not suppressed by treatment with cyclosporine and do not appear to depend on the elevation of left ventricular calcineurin activity.

Animals↗
PubMed1998

Type I collagen synthesis in cultured human fibroblasts: regulation by cell spreading, platelet-derived growth factor and interactions with collagen fibers.

Type I collagen protein and pro-alpha 1(I) collagen mRNA levels were investigated in human dermal fibroblasts cultured on substrates which induced distinct morphologies. Induction of type I collagen protein synthesis required cell spreading in monolayer cultures; mere attachment to dishes coated with 2-hydroxyethyl methacrylate (poly(HEMA)) did not suffice. Spread cells or round cells cultured on poly(HEMA) differed in collagen type I production, but pro-alpha 1(I) collagen mRNA levels were similar. Recombinant human platelet-derived growth factor (PDGF)-BB could replace cell spreading as a stimulus for collagen synthesis in cells cultured on poly(HEMA). At later time points, pro-alpha 1(I) collagen mRNA levels were down-regulated, although relatively less than type I collagen synthesis. Type I collagen synthesis by fibroblasts cultured in three-dimensional collagen gels was strongly down-regulated at both the protein and RNA levels. In addition to its capacity to stimulate collagen synthesis, PDGF-BB induced elongation and the formation of long processes by fibroblasts cultured in collagen gels. The stimulatory effect by cell spreading and PDGF-BB on collagen synthesis was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. However, inhibition of PI3K only inhibited induction of collagen synthesis by actively spreading cells or by PDGF-BB and did not induce a down-regulation of collagen synthesis in cells which had already spread. These data demonstrate that type I collagen protein synthesis is partly independent of pro-alpha 1(I) collagen mRNA levels but highly regulated by cell shape, although this could be decoupled by PDGF-BB. Both cell shape- and PDGF-BB-induced stimulation of collagen type I synthesis depends on a signalling pathway involving PI3K. Furthermore, levels of pro-alpha 1(I) collagen mRNA in fibroblasts are partly cell shape independent but are down-regulated by fibroblast interactions with native collagen fibers.

Adult↗
PubMed1997

The effects of angiotensin II and specific angiotensin receptor blockers on embryonic cardiac development and looping patterns.

The role of angiotensin II (Ang II) in the early embryonic development of the heart has not been examined. We have used RT-PCR to identify mRNA for angiotensinogen, angiotensin-converting enzyme, and the Ang II AT1 and AT2 receptors in embryonic day 10.25 Sprague-Dawley rats, and have used confocal microscopy to localize the AT1 receptor to the greater curvature of the developing ventricle in these animals at embryonic days (ED) 9.25 and 10.25. The antibodies used in immunolocalization studies did not distinguish between the AT1a and AT1b receptor subtypes. In whole embryo culture, Ang II added to the culture media resulted in increased ventricular growth and myocyte hypertrophy when treated embryos were compared to cultured littermate controls. Use of Losartan and PD123,319 to block the Ang II AT1 and AT2 receptors resulted in reduced ventricular development and cardiac dilation when compared to control and Ang II-treated embryos. Addition of Ang II and PD123,319 to the culture media also resulted in cardiac loop inversions which may be associated with disruption of normal myofibrillar development. These results clearly indicate an important role for Ang II in the early embryonic development of the heart.

Actins↗
PubMed1997

Analysis of heart development in cultured rat embryos.

The long-range goal of this research is to establish an in vitro system that will permit pertubation of mammalian heart development and in situ examination of the cellular and molecular events underlying cardiac morphogenesis. Rat embryos at 9.5-11.5 days of gestation were placed in culture bottles containing rat serum and Tyrode's solution. Embryos cultured for 24 and 48 h were compared to age-matched in vivo controls for morphological score, morphometric analysis of heart development, and confocal and electron microscopic analysis of myofiber pattern formation. Morphological scores indicated that embryos cultured for 24 h from day 9.5 to 10.5 had essentially normal development when compared to age-matched embryos allowed to develop in vivo. Development of embryos maintained for 48 h in culture was slightly delayed at 66-68% of age matched in vivo embryos. Analysis of hearts from embryos allowed to develop 9.5-11.5 days in vivo plus 24 and 48 h in culture showed that the ventricular thickness and height, as well as the truncal, atrial and ventricular diameters were equivalent to those of hearts from age-matched in vivo controls. Hearts from embryos allowed to develop from 11.5-12.5 days in vitro and cultured for 24 and 48 h had smaller left ventricular and atrial dimensions than controls. Cardiac myofibrillogenesis and myofibrillar pattern formation in embryos cultured from 9.5 days of in vivo development for 48 h were also normal. These studies indicate that the rat whole embryo culture system is a useful model to study several critical periods in mammalian heart development.

Animals↗
PubMed1997

Vinculin is an essential component for normal myofibrillar arrangement in fetal mouse cardiac myocytes.

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.

Animals↗
PubMed1997

Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes.

To determine whether the formation and maintenance of focal adhesions and costameres in cardiac myocytes are influenced by the mechanical forces that they transmit, we mechanically unloaded these cells by inhibiting their spontaneous contractile activity with the calcium-channel blocker nifedipine (12 microM). Interference-reflection and fluorescence microscopy revealed that within 24 h of arrest, beta 1-integrin- and vinculin-positive focal adhesions and costameres were disrupted. Loss of mature beta 1-integrin from the cell surface was observed in cell surface-labeling experiments and in Western blots. Subjecting nonbeating cells to a 5% static stretch for 24 h resulted in an increase of 21% for beta 1-integrin and 39% for vinculin. Stretching beating cells resulted in 71 and 9% increases, respectively. Intracellular concentrations of pre-beta 1 were not affected by contractile activity or by stretch. Our results indicate that mechanical forces stabilize the cellular levels of beta 1-integrin and vinculin by possibly regulating their association with the formation and maintenance of focal adhesions and costameres.

Animals↗
PubMed1996

Local and regional variations in myofibrillar patterns in looping rat hearts.

BACKGROUND: In chickens, cytodifferentiation, right side dominance in myofibril development, and variations in myofibrillar patterns in different areas and layers of the myocardial wall exist which have been implicated in the process of heart looping. Little comparable information is available for developing myofibrillar patterns in the early development of mammalian hearts. METHODS: We have used transmission electron microscopy (TEM), confocal scanning laser microscopy (CSLM), and 3-D reconstruction techniques also present in the looping hearts of embryonic day (ED) 9.5 to 11.5 rat hearts. RESULTS: Local and regional variations and right side dominance in myofibrillar patterns were shown during looping in 9.5 through 11.5 days of development in embryonic rat heart. At 9.5 days of development, myofibrils near the lumen of the myocardial wall were primarily in circumferential bands while near the pericardial surface they were primarily in longitudinal bands. In older embryos, regional variations in myofibrillar organization was found in areas associated with the cardiac cushions, trabeculae, and myocardial wall of the developing heart chambers. Based on sarcomeric structure, myofibrils in the ventricle and outflow tract were more advanced than those found in the atrial wall. CONCLUSIONS: The local and regional patterns of myofibrils in looping rat hearts are similar to those which have been found in developing chicken hearts. This study and others indicate cytodifferentiation and development of the contractile apparatus has a crucial role in the process of heart looping.

Animals↗
PubMed1996

Mechanical regulation of cardiac myocyte protein turnover and myofibrillar structure.

Mechanical forces play an essential role in regulating the synthesis and assembly of contractile proteins into the sarcomeres of cardiac myocytes. To examine if physical forces might also regulate the turnover of contractile proteins at a posttranslational site of control, beating and nonbeating neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. The L-type calcium channel blocker nifedipine (12 microM) was used to inhibit contraction. Pulse-chase biosynthetic labeling experiments demonstrated that contractile arrest accelerated the loss of isotopic tracer from the total myofibrillar protein fraction, myosin heavy chain (MHC), and actin, but not desmin. Myofibrillar abnormalities developed in parallel with these metabolic changes. A 5% static load appeared to partially stabilize myofibrillar structure in nonbeating NCM and suppressed the loss of isotopic tracer from the total myofibrillar protein fraction, MHC, and actin in beating and nonbeating NCM. Contractile activity and/or a static stretch promoted the accumulation of MHC, actin, and desmin. Applying a static load to myocytes that lacked preexisting myofibrils did not promote the assembly of sarcomeres or alter protein turnover. These data indicate that the turnover of MHC and actin is correlated with the organizational state of the myofibrillar apparatus.

Animals↗
PubMed1996

Exercise- and hypertension-induced collagen changes are related to left ventricular function in rat hearts.

Chronic hypertension, known to affect the collagen profile of the heart, and exercise result in impaired or improved heart function, respectively. Collagen types I [alpha 1(I)2 and alpha 2(I)] and III [alpha 1(III)3] are the predominant interstitial collagens thought to influence cardiac function, and the ratio of type III to I (collagen III/I) is thought to be a significant factor in the altered relaxation observed in hypertrophy. The present study tested the hypothesis that the myocardial structure and function are different in chronically exercise-trained vs. hypertensive rat hearts. Male rats were either chronically exercised (XTr) or submitted to experimental hypertension by coarctation of the abdominal aorta (Hyp) for 10 wks. Heart rate, blood pressure, and maximal rate of fall of the left ventricular pressure (-dp/dt) were recorded during isoproterenol stimulation. Results showed that both Hyp and XTr had higher heart weight and left ventricular weight-to-body weight ratios (P < 0.05). Mean arterial pressure (MAP) was higher in Hyp and lower in XTr (P < 0.05), whereas (-dP/dt)/MAP was diminished in Hyp but enhanced in XTr. Left ventricular collagen was higher in Hyp than XTr, whereas collagen III/I was reduced in Hyp compared with XTr (P < 0.05). Scanning and transmission electron microscopy also supported an accumulation of left ventricular collagen in Hyp compared with XTr. A negative correlation was observed between collagen III/I and (-dP/dt)/ MAP (r = -0.91; P < 0.05). These results suggest an important relationship between adaptations in left ventricular collagen and the changes in diastolic function observed in both chronic hypertension and exercise cardiac stress.

Animals↗