Effects of biglycan deficiency on myocardial infarct structure and mechanics

Biglycan, a small leucine-rich proteoglycan, has been shown to interact with extracellular matrix (ECM) collagen and may influence fibrillogenesis. We hypothesized that biglycan contributes to post-myocardial infarction (MI) scar development and that the absence of biglycan would result in altered scar structure and mechanics. Anterior MI was induced in biglycan hemizygous null and wild-type mice by permanent ligation of the left coronary artery. The initial extent of ischemic injury was similar in the two groups, as was the infarct size after 30 days, although there was some tendency toward reduced expansion in the biglycan-null. Electron microscopy revealed that collagen fibrils had a smaller average diameter and a narrower range in the biglycan-null scar, as well as appearing more densely packed. In vivo strain analysis showed that biglycan-null scars were stiffer than the wild-type. Remote LV collagen concentration tended to be reduced in biglycan-null hearts, but the difference was not statistically significant. Null-expression of biglycan may alter collagen fibril ultrastructure, and thereby influence scar mechanics and remodeling.     

Extracellular matrix remodeling in canine and mouse myocardial infarcts

Extracellular matrix proteins not only provide structural support, but also modulate cellular behavior by activating signaling pathways. Healing of myocardial infarcts is associated with dynamic changes in the composition of the extracellular matrix; these changes may play an important role in regulating cellular phenotype and gene expression. We examined the time course of extracellular matrix deposition in a canine and mouse model of reperfused infarction. In both models, myocardial infarction resulted in fragmentation and destruction of the cardiac extracellular matrix, extravasation of plasma proteins, such as fibrinogen and fibronectin, and formation of a fibrin-based provisional matrix providing the scaffold for the infiltration of granulation tissue cells. Lysis of the plasma-derived provisional matrix was followed by the formation of a cell-derived network of provisional matrix composed of cellular fibronectin, laminin, and hyaluronic acid and containing matricellular proteins, such as osteopontin and osteonectin/SPARC. Finally, collagen was deposited in the infarct, and the wound matured into a collagen-based scar with low cellular content. Although the canine and mouse infarcts exhibited a similar pattern of extracellular matrix deposition, deposition of the provisional matrix was more transient in the mouse infarct and was followed by earlier formation of a mature collagen-based scar after 7-14 days of reperfusion; at the same timepoint, the canine infarct was highly cellular and evolving. In addition, mature mouse infarcts showed limited collagen deposition and significant tissue loss leading to the formation of a thin scar. In contrast, dogs exhibited extensive collagen accumulation in the infarcted area. These species-specific differences in infarct wound healing should be taken into account when interpreting experimental infarction studies and when attempting to extrapolate the findings to the human pathological process.     

CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response

Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of "stop signals" that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44(-/-) animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44(null) infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44(null) cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-beta in comparison to wild-type fibroblasts. The healing defects in CD44(-/-) mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.     

ANG II type 1A receptor signaling causes unfavorable scar dynamics in the postinfarct heart

Blockade of ANG II type 1A receptor (AT(1A)) is known to attenuate postinfarction [postmyocardial infarction (post-MI)] heart failure, accompanying reduction in fibrosis of the noninfarcted area. In the present study, we investigated the influence of AT(1A) blockade on the infarcted tissue itself. Consistent with earlier reports, AT(1A) knockout (AT(1A)KO) mice showed significantly attenuated left ventricular (LV) remodeling (dilatation) and dysfunction compared with wild-type (WT) mice. Morphometry revealed that the infarcted wall was thicker and had a smaller circumferential length in AT(1A)KO than WT hearts. In addition, significantly greater numbers of cells were present within infarcts in AT(1A)KO hearts 4 wk post-MI; most notably, there was an abundance of vessels and myofibroblasts. One week post-MI, the incidence of apoptosis among granulation tissue cells was fewer (3.3 +/- 0.4 vs. 4.4 +/- 0.5% in WT, P < 0.05), whereas vessel proliferation was higher in AT(1A)KO hearts, which likely explains the later abundance of cells within the scar tissue. Insulin-like growth factor receptor-I was upregulated and its downstream signal protein kinase B (Akt) was significantly activated in infarcted AT(1A)KO hearts compared with WT hearts. Inactivation of Akt with wortmannin partially but significantly prevented the benefits observed in AT(1A)KO. Collectively, in AT(1A)KO hearts, Akt-mediated granulation tissue cell proliferation and preservation resulting from antiapoptosis likely contributed to an abundant cell population that altered the infarct scar structure, thereby reducing wall stress and attenuating LV dilatation and dysfunction at the chronic stage. In conclusion, altered structural dynamics of infarct scar and increasing myocardial fibrosis may be responsible for the deleterious effects of AT(1A) signaling following MI.     

Two‐photon microscopy of healthy,infarcted and stem‐cell treated regenerating heart

Two‐photon excitation autofluorescence (produced in myocytes) and second‐harmonic generation (produced mainly by collagen) allow label‐free visualization of these two important components of myocardium. Because of their different emission wavelengths, these two signals can be separated spectrally. Here, we examine two‐photon microscopy images of healthy, infarcted and stem‐cell treated rat hearts. We find that in infarcted heart, regions distant from the site of infarct are similar to healthy tissue in composition (mostly myocytes, very little collagen) and organization (densely packed myocytes), but infarct regions are characterized by sparse myocytes and high collagen content indicative of scar tissue formation. Stem cell treated hearts, in contrast, show regions of intertwined myocytes and collagen throughout the infarct, suggesting reduced tissue damage. Finally, these results offer interesting insights into our ongoing polarized light studies of cardiac tissue anisotropy, and reveal that both tissue composition and tissue micro‐organization are reflected in polarization‐measured linear retardance values. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The plating of rat scar myofibroblasts on matrigel unmasks a novel phenotype; the self assembly of lumen-like structures

During tissue healing, the primary role of myofibroblasts involves the synthesis and deposition of collagen. However, it has also been reported that selective populations of myofibroblasts can acquire the phenotype and/or differentiate to other cells types. The present study tested the hypothesis that myofibroblasts isolated from the scar of the ischemically damaged rat heart can recapitulate an endothelial cell-like response when plated in a permissive in vitro environment. Scar myofibroblasts, neonatal and adult ventricular fibroblasts express smooth muscle α-actin, collagen α(1) type 1 and a panel of pro-fibrotic and pro-angiogenic peptide growth factor mRNAs. Myofibroblasts plated alone on matrigel led to the self assembly of lumen-like structures whereas neonatal and adult rat ventricular fibroblasts were unresponsive. Myofibroblasts labeled with the fluorescent cell tracker CM-DiI were injected in the viable myocardium of 3-day post-myocardial infarcted Sprague-Dawley rats and sacrificed 7 days later. Injected CM-DiI-labeled myofibroblasts were detected predominantly in the peri-infarct/infarct region, highlighting their migration to the damaged region. However, engrafted myofibroblasts in the peri-infarct/infarct region were unable to adopt an endothelial cell-like phenotype or lead to the de novo formation of CM-DiI-labeled blood vessels. The non-permissive nature of the infarct region may be attributed at least in part to the presence of growth-promoting stimuli as TGF-β and the β-adrenergic agonist isoproterenol inhibited the self assembly of lumen-like structures by myofibroblasts. Thus, when plated in a permissive in vitro environment, scar myofibroblasts can self assemble and form lumen-like structures providing an additional novel phenotype distinguishing this population from normal ventricular fibroblasts.     

Angiotensin II type 1 receptor regulation and differential trophic effects on rat cardiac myofibroblasts after acute myocardial infarction

Fibroblast growth in the scar and surviving tissue is a key element of the remodeling post myocardial infarction. The regulation of fibroblast growth after acute myocardial infarction remains to be determined. Recently, Angiotensin II has been demonstrated to be a mitogen for neonatal cardiac fibroblasts. In this study adult rat cardiac fibroblasts were isolated from different regions of the infarcted rat heart and Angiotensin II effects examined. Adult Wistar-rats were sham operated or left coronary artery ligated. After 4 days, hearts were removed and fibroblasts from sham operated, infarct- and non-infarct regions of the left ventricle isolated. Radioligand binding studies were performed and cell number, cell area, total protein, and AT(1) receptor mRNA after stimulation determined. Radioligand binding studies demonstrated that myofibroblasts expressed a single class of high affinity Angiotensin II AT(1) receptors. Myofibroblasts from the infarct area revealed a lower maximal binding capacity, compared to sham operated myocardium. Conversely, myofibroblasts from the non-infarct area had a higher expression of Angiotensin II AT(1) receptor mRNA compared to sham operated myofibroblasts. Angiotensin II (1 microM, 48 h) increased cell-number in sham operated and non-infarct, but not in infarct myofibroblasts. Angiotensin II elevated total protein in sham operated, non-infarct, and infarct myofibroblasts. In addition, Angiotensin II increased cell area in sham operated and infarct myofibroblasts. These data demonstrate that Angiotensin II acted as a mitogen in sham operated and non-infarct myofibroblasts and stimulated hypertrophy in infarct myofibroblasts. These regional different effects of Angiotensin II might participate in the remodeling post myocardial infarction.     

Diabetes influences cardiac extracellular matrix remodelling after myocardial infarction and subsequent development of cardiac dysfunction

This study was conducted to examine the influence of acute streptozotocin‐induced diabetes on cardiac remodelling and function in mice subjected to myocardial infarction (MI) by coronary artery ligation. Echocardiography analysis indicated that diabetes induced deleterious cardiac functional changes as demonstrated by the negative differences of ejection fraction, fractional shortening, stroke volume, cardiac output and left ventricular volume 24 hrs after MI. Temporal analysis for up to 2 weeks after MI showed higher mortality in diabetic animals because of cardiac wall rupture. To examine extracellular matrix remodelling, we used fluorescent molecular tomography to conduct temporal studies and observed that total matrix metalloproteinase (MMP) activity in hearts was higher in diabetic animals at 7 and 14 days after MI, which correlated well with the degree of collagen deposition in the infarct area visualized by scanning electron microscopy. Gene arrays indicated temporal changes in expression of distinct MMP isoforms after 1 or 2 weeks after MI, particularly in diabetic mice. Temporal changes in cardiac performance were observed, with a trend of exaggerated dysfunction in diabetic mice up to 14 days after MI. Decreased radial and longitudinal systolic and diastolic strain rates were observed over 14 days after MI, and there was a trend towards altered strain rates in diabetic mouse hearts with dyssynchronous wall motion clearly evident. This correlated with increased collagen deposition in remote areas of these infarcted hearts indicated by Masson's trichrome staining. In summary, temporal changes in extracellular matrix remodelling correlated with exaggerated cardiac dysfunction in diabetic mice after MI.     

Decreased Smad 7 expression contributes to cardiac fibrosis in the infarcted rat heart

We examined the role of the transforming growth factor (TGF)-beta(1) signaling inhibitor Smad 7 in cardiac fibrosis. TGF-beta(1) (10 ng/ml) was found to increase cytosolic Smad 7 expression in primary adult rat fibroblasts and induce rapid nuclear export of exogenous Smad 7 in COS-7 cells. Furthermore, overexpression of Smad 7 in primary adult fibroblasts was associated with suppressed collagen type I and III expression. We detected Smad 7, phosphorylated Smad 2, TGF-beta type I receptor (TbetaRI), and TGF-beta(1) proteins in postmyocardial infarct (MI) rat hearts. In 2 and 4 wk post-MI hearts, Smad 7 and TbetaRI expression were decreased in scar tissue, whereas TGF-beta(1) expression was increased in scar and viable tissue. In the 8 wk post-MI heart, Smad 7 expression was decreased in both scar tissue and myocardium remote to the infarct scar. Finally, we confirmed that these changes are paralleled by decreased expression of cytosolic phosphorylated receptor-regulated Smad 2 in 4-wk viable myocardium and in 2- and 4-wk infarct scar tissues. Taken together, our data imply that decreased inhibitory Smad 7 signal in cardiac fibroblasts may play a role in the pathogenesis of cardiac fibrosis in the post-MI heart.     

Residual strain in ischemic ventricular myocardium.

Structural remodeling during acute myocardial infarction affects ventricular wall stress and strain. To see whether acute myocardial infarction alters residual stress and strain in the left ventricle (LV), we measured opening angles in rat hearts after 30 minutes of left coronary artery occlusion. The mean opening angle in 18 ischemic hearts (51 +/- 20 deg) was significantly greater than in five sham-operated controls (29 +/- 11 deg, P < 0.05). To determine whether these alterations in residual strain may be associated with strain softening caused by systolic overstretch of the noncontracting ischemic tissue, we also measured opening angles in isolated hearts that had been passively inflated to high LV pressures (120 mmHg). The mean opening angle of the strain-softened hearts was not significantly different from the sham-operated hearts (34 +/- 27 deg, P = 0.74). Mean collagen area fractions in the myocardium were not significantly different between ischemic hearts (0.027 +/- 0.014) and the nonischemic group (0.022 +/- 0.011). Although there were significant differences in opening angles measured with ischemia, they do not appear to be a result of altered extracellular collagen content or softening associated with overstretch. Thus, there is a significant change in residual strain associated with acute ischemia that may be related to changes in collagen fiber structure, myocyte structure, or metabolic state.     

A multi-station culture force monitor system to study cellular contractility

Cellular contraction contributes to the formation of scar tissue, which is characterized by an over-produced, disorganized collagen matrix. To study the contractility of cells in vitro and its potential contribution to scar tissue formation, we have developed a multi-station culture force monitor (CFM) system. This system consists of four vertical cantilever beams with semiconductor strain gages and a computerized data acquisition unit to monitor contractile forces of the cells in a collagen gel. Calibration showed that this system has a highly linear voltage-force relationship (R(2) > 0.99). Further, to demonstrate the applicability of this system, contractile forces of human skin fibroblasts in a collagen gel were measured. These fibroblasts were found to produce an average force of 0.2 nN/cell, which is consistent with the data in literature. The significant advantage of this CFM system is its ability to test multiple samples simultaneously. Therefore, the system can facilitate statistical design and analysis of experiments to study the effects of growth factors (e.g., TGF-betas) on cellular contraction and their potential role in scar tissue formation.     

Differences between scar and dermal cultured fibroblasts derived from a patient with recurrent abdominal incision wound herniation.

Fibroblasts were derived from dermis and scar of a 47-year-old white man with a recurrent incisional hernia as a result of fractured ribs. The scar was thin and stretched, suggesting a defect in the maturation of granulation tissue. After surgical repair, biopsy specimens of discarded scar and skin were used to generate fibroblast cell lines. Fibroblasts maintained in medium containing 10% fetal bovine serum and antibiotic were studied between their third and eighth passage. By phase contrast microscopy, no structural differences were obvious, but it was noted that to pass scar fibroblasts, a more aggressive trypsin regimen was required. Immunohistologic and Western blot analysis of patient scar fibroblasts showed (1) more a smooth muscle actin within stress fibers, (2) increased expression of the vitronectin integrin receptor alpha(v) (CD 51), and (3) reduced expression of the collagen integrin receptor alpha2 (CD 49b). The expression of vinculin from focal adhesions or a tubulin from microtubules was the same among cell lines. Contractions of scar and dermal fibroblast-populated collagen lattice were compared. At 24 hours, contractions were 69 percent with newborn fibroblasts (normal); 68 percent for patient dermal fibroblasts; and only 48 percent for patient scar fibroblasts. The retarded contraction of scar fibroblast-populated collagen lattice was significant (p > or = 0.002). Myosin ATPase activity, critical for lattice contraction, and cell migration were equivalent among all cell lines. A plausible mechanism for the retardation of scar lattice contraction is disruption of fibroblasts and collagen interactions, for which the attachment of cells to collagen is altered. It is proposed that either the decrease in the expression of collagen integrin receptor alpha2 (CD 49b), an increase in the expression of the vitronectin receptor alpha(v) (CD 51), or a combination of both is responsible for disruption of collagen fibroblast interactions.     

Systematic Characterization of Myocardial Inflammation,Repair, and Remodeling in a Mouse Model of Reperfused Myocardial Infarction

Mouse models of myocardial infarction are essential tools for the study of cardiac injury, repair, and remodeling. Our current investigation establishes a systematic approach for quantitative evaluation of the inflammatory and reparative response, cardiac function, and geometry in a mouse model of reperfused myocardial infarction. Reperfused mouse infarcts exhibited marked induction of inflammatory cytokines that peaked after 6 hr of reperfusion. In the infarcted heart, scar contraction and chamber dilation continued for at least 28 days after reperfusion; infarct maturation was associated with marked thinning of the scar, accompanied by volume loss and rapid clearance of cellular elements. Echocardiographic measurements of end-diastolic dimensions correlated well with morphometric assessment of dilative remodeling in perfusion-fixed hearts. Hemodynamic monitoring was used to quantitatively assess systolic and diastolic function; the severity of diastolic dysfunction following myocardial infarction correlated with cardiomyocyte hypertrophy and infarct collagen content. Expression of molecular mediators of inflammation and cellular infiltration needs to be investigated during the first 72 hr, whereas assessment of dilative remodeling requires measurement of geometric parameters for at least four weeks after the acute event. Rapid initiation and resolution of the inflammatory response, accelerated scar maturation, and extensive infarct volume loss are important characteristics of infarct healing in mice.     

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re‐establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full‐length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9‐week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell‐based gene therapy provides a new approach to cardiac regeneration.     

Postinfarction healing dynamics in the mechanically unloaded rat left ventricle

The healing process is a key determinant for postinfarction left ventricular (LV) remodeling and the development of heart failure, which could be influenced by mechanical (pressure and/or volume) load. So far, limited information exists regarding an indepth characterization of the postinfarct healing process in the mechanically unloaded state. In the present work, we performed isogenic Lewis-to-Lewis rat abdominal heterotopic heart transplantation, which is characterized by hemodynamic unloading in the left ventricle, and simultaneously ligated the left anterior descending coronary artery (T-infarct group). Pathological evolution was dynamically compared with that of in situ infarcted Lewis hearts (I-infarct group) on days 3, 7, 14, and 35. There was a remarkable myocardial salvage in the unloaded heart, as shown by the improvement in infarct size (T-infarct group: 25.47% ± 4.31% vs. I-infarct group: 38.46% ± 4.82%, P < 0.01) and the smaller fraction of fibrosis in infarct segments (T-infarct group: 42.12% ± 8.40% vs. I-infarct group: 75.65% ± 10.51%, P < 0.01). In addition, there was a progressive disorganization of the two-dimensional collagen fiber alignment as well as retarded collagen fiber maturation in the T-infarct group. We also observed enhanced angiogenesis, lymphangiogenesis, and inflammatory cell retention in the infarct region during mechanical unloading. Moreover, capillary density and collagen deposition were significantly increased in the noninfarcted area of the unloaded heart compared with the same region in the in situ infarcted heart. In conclusion, ischemic insult in the mechanically unloaded heart elicits an altered inflammatory and healing response, which is characterized by myocardial salvage, delayed resolution of inflammation, and disorganization of the collagen orientation in the infarcted region. These findings could provide novel insights into the contribution of hemodynamic load in the postinfarction healing process. Further studies are warranted to elucidate its potential mechanism.     

Cardiotrophin-1: Expression in experimental myocardial infarction and potential role in post-MI wound healing

Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been shown to be elevated in the serum of patients with ischemic heart disease and valvular heart disease, and induces cardiomyocyte hypertrophy in vitro. We investigated expression of CT-1 in post-MI rat heart and the effect of CT-1 on cultured primary adult rat cardiac fibroblasts. Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak1, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of Jak/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased ³H-leucine and ³H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (P1CP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. Our results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis. Further work is required to determine factors that induce CT-1 expression and interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure.     

Dynamic protrusive cell behaviour generates force and drives early matrix contraction by fibroblasts

We investigated the cellular mechanisms underlying force generation and matrix contraction, using human corneal, Tenon's and scleral fibroblasts in a standard collagen matrix. We used timelapse light and confocal reflection microscopy to analyse concomitantly cell behaviour and matrix remodeling during contraction and devised a novel index to quantify dynamic cell behaviour in 3D. Based on the previously described culture force monitor, a novel simultaneous imaging and micro-culture force monitor system (SIM-CFM) was developed to measure the mechanical strain generated during matrix contraction whilst simultaneously recording cell and matrix behaviour. Ocular fibroblasts show marked differences in macroscopic matrix contraction profiles, with corneal fibroblasts inducing the strongest, and scleral the weakest, contraction. We identified four factors that determine the early matrix contraction profile: 1) cell size, 2) intrinsic cellular force, 3) dynamic cell protrusive activity and 4) net pericellular matrix displacement. Intrinsic cellular force and dynamic activity appear to be independent unique characteristics of each cell type and might serve as predictors of matrix contraction. The identification of these factors raises the fundamental new possibilities of predicting the ability of tissues to contract and scar and of modulating tissue contraction by targeting intracellular pathways linked to protrusive activity and force generation.     

Age-related differences in postinfarct left ventricular rupture and remodeling

Cardiac rupture is more prevalent in elderly patients with first onset of acute myocardial infarct (MI), but the mechanism remains unexplored. We investigated the differences in the incidence of cardiac rupture and early left ventricular (LV) remodeling following coronary artery ligation between old (12-mo) and young (3-mo) C57Bl/6 male mice and explored responsible mechanisms. The incidence of rupture within 1 wk after MI was significantly higher in old than in young mice (40.7 vs. 18.3%, P = 0.013) despite a similar infarct size in both age groups. Old mice dying of rupture had more severe infarct expansion than young counterparts. Echocardiography and catheterization at day 7 revealed more profound LV chamber dilatation and dysfunction as well as higher blood pressures in aged mice. At day 3 after MI immediately before the peak of rupture occurrence, we observed significantly higher content of type I and III collagen, a greater density of macrophage and neutrophil, and markedly enhanced mRNA expression of inflammatory cytokines in the infarcted myocardium in old than in young mice. Furthermore, a more dramatic increment of matrix metalloproteinase (MMP)-9 activity was found in old than in young infarcted hearts, in keeping with enhanced inflammatory response. Collectively, these results revealed that old mice had a higher risk of post-MI cardiac rupture despite a higher level of collagen content and cross-linking. Enhanced inflammatory response and subsequent increase in MMP-9 activity together with higher blood pressure are important factors responsible for the higher risk of cardiac rupture and more severe LV remodeling in the aged heart following acute MI.     

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.     

Extracellular matrix (ECM) microstructural composition regulates local cell-ECM biomechanics and fundamental fibroblast behavior: a multidimensional perspective.

The extracellular matrix (ECM) provides the principal means by which mechanical information is communicated between tissue and cellular levels of function. These mechanical signals play a central role in controlling cell fate and establishing tissue structure and function. However, little is known regarding the mechanisms by which specific structural and mechanical properties of the ECM influence its interaction with cells, especially within a tissuelike context. This lack of knowledge precludes formulation of biomimetic microenvironments for effective tissue repair and replacement. The present study determined the role of collagen fibril density in regulating local cell-ECM biomechanics and fundamental fibroblast behavior. The model system consisted of fibroblasts seeded within collagen ECMs with controlled microstructure. Confocal microscopy was used to collect multidimensional images of both ECM microstructure and specific cellular characteristics. From these images temporal changes in three-dimensional cell morphology, time- and space-dependent changes in the three-dimensional local strain state of a cell and its ECM, and spatial distribution of beta1-integrin were quantified. Results showed that fibroblasts grown within high-fibril-density ECMs had decreased length-to-height ratios, increased surface areas, and a greater number of projections. Furthermore, fibroblasts within low-fibril-density ECMs reorganized their ECM to a greater extent, and it appeared that beta1-integrin localization was related to local strain and ECM remodeling events. Finally, fibroblast proliferation was enhanced in low-fibril-density ECMs. Collectively, these results are significant because they provide new insight into how specific physical properties of a cell's ECM microenvironment contribute to tissue remodeling events in vivo and to the design and engineering of functional tissue replacements.