Inflammatory and regenerative processes in bioresorbable synthetic pulmonary valves up to two years in sheep-Spatiotemporal insights augmented by Raman microspectroscopy.

In situ heart valve tissue engineering is an emerging approach in which resorbable, off-the-shelf available scaffolds are used to induce endogenous heart valve restoration. Such scaffolds are designed to recruit endogenous cells in vivo, which subsequently resorb polymer and produce and remodel new valvular tissue in situ. Recently, preclinical studies using electrospun supramolecular elastomeric valvular grafts have shown that this approach enables in situ regeneration of pulmonary valves with long-term functionality in vivo. However, the evolution and mechanisms of inflammation, polymer absorption and tissue regeneration are largely unknown, and adverse valve remodeling and intra- and inter-valvular variability have been reported. Therefore, the goal of the present study was to gain a mechanistic understanding of the in vivo regenerative processes by combining routine histology and immunohistochemistry, using a comprehensive sheep-specific antibody panel, with Raman microspectroscopy for the spatiotemporal analysis of in situ tissue-engineered pulmonary valves with follow-up to 24 months from a previous preclinical study in sheep. The analyses revealed a strong spatial heterogeneity in the influx of inflammatory cells, graft resorption, and foreign body giant cells. Collagen maturation occurred predominantly between 6 and 12 months after implantation, which was accompanied by a progressive switch to a more quiescent phenotype of infiltrating cells with properties of valvular interstitial cells. Variability among specimens in the extent of tissue remodeling was observed for follow-up times after 6 months. Taken together, these findings advance the understanding of key events and mechanisms in material-driven in situ heart valve tissue engineering. STATEMENT OF SIGNIFICANCE: This study describes for the first time the long-term in vivo inflammatory and regenerative processes that underly in situ heart valve tissue engineering using resorbable synthetic scaffolds. Using a unique combinatorial analysis of immunohistochemistry and Raman microspectroscopy, important spatiotemporal variability in graft resorption and tissue formation was pinpointed in in situ tissue-engineered heart valves, with a follow-up time of up to 24 months in sheep. This variability was correlated to heterogenous regional cellular repopulation, most likely instigated by region-specific differences in surrounding tissue and hemodynamics. The findings of this research contribute to the mechanistic understanding of in situ tissue engineering using resorbable synthetics, which is necessary to enable rational design of improved grafts, and ensure safe and robust clinical translation.

An initial evaluation of analyser-based phase-contrast X-ray imaging of carotid plaque microstructure.

Carotid artery plaque instability can result in rupture and lead to ischaemic stroke. Stability of plaques appears to be a function of composition. Current non-invasive imaging techniques are limited in their ability to classify distinct histological regions within plaques. Phase-contrast (PC) X-ray imaging methods are an emerging class of techniques that have shown promise for identifying soft-tissue features without use of exogenous contrast agents. This is the first study to apply analyser-based X-ray PC imaging in CT mode to provide three-dimensional (3D) images of excised atherosclerotic plaques. The results provide proof of principle for this technique as a promising method for analysis of carotid plaque microstructure. Multiple image radiography CT (MIR-CT), a tomographic implementation of X-ray PC imaging that employs crystal optics, was employed to image excised carotid plaques. MIR-CT imaging yields three complementary images of the plaque's 3D X-ray absorption, refraction and scatter properties. These images were compared with histological sections of the tissue. X-ray PC images were able to identify the interface between the plaque and the medial wall. In addition, lipid-rich and highly vascularized regions were visible in the images as well as features depicting inflammation. This preliminary research shows MIR-CT imaging can reveal details about plaque structure not provided by traditional absorption-based X-ray imaging and appears to identify specific histological regions within plaques. This is the first study to apply analyser-based X-ray PC imaging to human carotid artery plaques to identify distinct soft-tissue regions.

Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves.

BACKGROUND: The mechanisms of extracellular matrix changes accompanying myxomatous valvular degeneration are uncertain. METHODS AND RESULTS: To test the hypothesis that valvular interstitial cells mediate extracellular matrix degradation in myxomatous degeneration by excessive secretion of catabolic enzymes, we examined the functional characteristics of valvular interstitial cells in 14 mitral valves removed for myxomatous degeneration from patients with mitral regurgitation and in 11 normal mitral valves obtained at autopsy. Immunohistochemical staining assessed (1) cell phenotype using antibodies to alpha-actin (microfilaments), vimentin and desmin (intermediate filaments), smooth muscle myosin (SM1), and SMemb (a nonmuscle myosin produced by activated mesenchymal cells) and (2) the expression of proteolytic activity using antibodies to collagenases (matrix metalloproteinase [MMP]-1, MMP-13), gelatinases (MMP-2, MMP-9), cysteine endoproteases (cathepsin S and K), and interleukin-1beta, a cytokine that can induce secretion of proteolytic enzymes. Although interstitial cells in normal valves stained positively for vimentin, but not alpha-actin or desmin, cells in myxomatous valves contained both vimentin and alpha-actin or desmin (characteristics of myofibroblasts). Moreover, cells in myxomatous valves strongly expressed SMemb, MMPs, cathepsins, and interleukin-1beta, which were weakly stained in controls. Nevertheless, interstitial cells in both groups strongly expressed procollagen-I mRNA (in situ hybridization), suggesting preserved ability to synthesize collagen in myxomatous valves. CONCLUSIONS: Interstitial cells in myxomatous valves have features of activated myofibroblasts and express excessive levels of catabolic enzymes, without altered levels of interstitial collagen mRNA. We conclude that valvular interstitial cells regulate matrix degradation and remodeling in myxomatous mitral valve degeneration.

Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.

Cardiac valves arise from endocardial cushions, specialized regions of the developing heart that are formed by an endothelial-to-mesenchymal cell transdifferentiation. Whether and to what extent this transdifferentiation is retained in mature heart valves is unknown. Herein we show that endothelial cells from mature valves can transdifferentiate to a mesenchymal phenotype. Using induction of alpha-smooth muscle actin (alpha-SMA), an established marker for this process, two distinct pathways of transdifferentiation were identified in clonally derived endothelial cell populations isolated from ovine aortic valve leaflets. alpha-SMA expression was induced by culturing clonal endothelial cells in medium containing either transforming growth factor-beta or low levels of serum and no basic fibroblast growth factor. Cells induced to express alpha-SMA exhibited markedly increased migration in response to platelet-derived growth factor-BB, consistent with a mesenchymal phenotype. A population of the differentiated cells co-expressed CD31, an endothelial marker, along with alpha-SMA, as seen by double-label immunofluorescence. Similarly, this co-expression of endothelial markers and alpha-SMA was detected in a subpopulation of cells in frozen sections of aortic valves, suggesting the transdifferentiation may occur in vivo. Hence, the clonal populations of valvular endothelial cells described here provide a powerful in vitro model for dissecting molecular events that regulate valvular endothelium.

Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo.

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.

Dilated cardiomyopathy in two transgenic mouse lines expressing activated G protein alpha(q): lack of correlation between phospholipase C activation and the phenotype.

We previously described a transgenic mouse line (alpha(q)*52) in which cardiac-specific expression of activated G alpha(q)protein (HA alpha(q)*) leads to activation of phospholipase C beta (PLC beta), the immediate downstream target of HA alpha(q)*, with subsequent development of cardiac hypertrophy and dilation. We now describe a second, independent line in the same genetic background (alpha(q)*44h) with lower expression of HA alpha(q)* protein that ultimately results in the same phenotype: dilated cardiomyopathy (DCM) with severely impaired left ventricular systolic function (assessed by M-mode and 2D echocardiography), but with a much delayed disease onset. We asked if PLC activation correlates with the development of the phenotype. At 12-14 months, 65% of alpha(q)*44h mice still had normal cardiac function and ventricular weight/body weight ratios (VW/BW). However, their basal PLC activity, which began to increase in ventricles at 6 months, was threefold higher than in wild-type by 12 months. This increase was even more pronounced than in 2.5-month-old alpha(q)*52 mice, in which a twofold increase was accompanied by a 25% increase in VW/BW. Furthermore, at 12-14 months the increase in PLC activity in alpha(q)*44h mice with and without DCM was comparable. Thus, the delayed time course in alpha(q)*44h mice unmasked a lack of correlation between PLC activation and development of DCM in response to HA alpha(q)* expression, suggesting a role for additional pathways and/or mechanisms. It also revealed a differential temporal regulation of protein kinase C isoform expression. The markedly different ages of disease onset in these two mouse lines provide a model for studying both genetic modifying factors and potential environmental influences in DCM.

Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis.

We previously showed that the expression of tenascin (TN-C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, and matrix metalloproteinase-2 (MMP-2) are coordinated and interdependent in cultured vascular smooth muscle cells. In this study, we hypothesized that TN-C and MMP-2 are mechanistically involved in the pathobiology of calcific aortic stenosis. Human calcific aortic stenosis cusps demonstrated immunohistochemically prominent deposition of TN-C, MMP-2, and alkaline phosphatase activity, as well as MMP-2 gelatinolytic activity. Although far lesser amounts of TN-C were noted in several of the grossly non-calcified valve cusps, MMP-2 and AP were never detected. Further, when aortic valve interstitial cells (both sheep and human) were cultivated on collagen supplemented with TN-C, both MMP-2 mRNA expression and MMP-2 gelatinolytic activity (both pro and active forms), were up-regulated compared to control. These observations support the view that accumulation of first TN-C and then MMP-2 are associated with progression of calcification. The residual presence of these proteins in severe calcifications is indicative of their involvement in the pathogenesis.

Comparison of two murine models of familial hypertrophic cardiomyopathy.

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro.

BACKGROUND: Unstable atherosclerotic plaques that cause acute coronary events usually contain abundant macrophages expressing matrix metalloproteinases (MMPs) and tissue factor (TF), molecules that probably contribute to plaque rupture and subsequent thrombus formation. Lipid lowering with HMG-CoA reductase inhibitors reduces acute coronary events. METHODS AND RESULTS: To test whether lipid lowering with an HMG-CoA reductase inhibitor retards macrophage accumulation in rabbit atheroma, we administered cerivastatin to immature Watanabe heritable hyperlipidemic rabbits (cerivastatin group, n=10, cerivastatin 0.6 mg x kg(-1) x d(-1); control group, n=9, saline 0.6 mL x kg(-1) x d(-1)) for 32 weeks and measured macrophage accumulation and expression of MMPs and TF. Serum cholesterol levels after 32 weeks were 809+/-40 mg/dL (control group) and 481+/-24 mg/dL (treated group). Cerivastatin diminished accumulation of macrophages in aortic atheroma. Macrophage expression of MMP-1, MMP-3, MMP-9, and TF also decreased with cerivastatin treatment. Cerivastatin reduced the number of macrophages expressing histone mRNA (a sensitive marker of cell proliferation) detected by in situ hybridization but did not alter macrophages bearing a marker of death (TUNEL staining). Cerivastatin treatment (>or=0.01 micromol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages. CONCLUSIONS: These results suggest that lipid lowering with HMG-CoA reductase inhibitors alters plaque biology by reducing proliferation and activation of macrophages, prominent sources of molecules responsible for plaque instability and thrombogenicity.

An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy.

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (alphaMHC(403/+)), when treated with calcineurin inhibitors or a K(+)-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca(2+) concentrations in wild-type cardiac myocytes; alphaMHC(403/+) myocytes failed to respond. Pretreatment with a Ca(2+)-channel antagonist abrogated diastolic Ca(2+) changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated alphaMHC(403/+) mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca(2+) responses that initiate a hypertrophic response. These data define an important Ca(2+)-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease.

Patch augmentation of the pulmonary artery with bioabsorbable polymers and autologous cell seeding.

OBJECTIVE: In recent years bioabsorbable synthetic or biologic materials have been used to augment the pulmonary artery or the right ventricular outflow tract. However, each of these polymers has one or more shortcomings. None of these patch materials has been seeded with cells. Thus, we have tested a fast-absorbing biopolymer, poly-4-hydroxybutyric acid, with autologous cell seeding for patch augmentation of the pulmonary artery in a juvenile sheep model. METHODS: Vascular cells were isolated from ovine peripheral veins (n = 6). Bioabsorbable porous poly-4-hydroxybutyric acid patches (porosity > 95%) were seeded on 3 consecutive days with a mixed vascular cell suspension (21.3 +/- 1.3 x 10(6) cells). Forty-five (+/- 2) days after the vessel harvest, 1 unseeded and 6 autologously seeded control patches were implanted into the proximal pulmonary artery. The animals received no postoperative anticoagulation. Follow-up was performed with echocardiography after 1 week and before explantation after 1, 7, and 24 weeks (2 animals each) for the seeded control patches and after 20 weeks for the nonseeded control patch. RESULTS: All animals survived the procedure. Postoperative echocardiography of the seeded patches demonstrated a smooth surface without dilatation or stenosis. Macroscopic appearance showed a smooth internal surface with increasing tissue formation. Histology at 169 days demonstrated a near-complete resorption of the polymer and formation of organized and functional tissue. Biochemical assays revealed increasing cellular and extracellular matrix contents. The control patch showed a slight bulging, indicating a beginning dilatation. CONCLUSION: This experiment showed that poly-4-hydroxybutyric acid is a feasible patch material in the pulmonary circulation.

Early in vivo experience with tissue-engineered trileaflet heart valves.

BACKGROUND: Tissue engineering is a new approach in which techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional autologous tissue. Workers at our laboratory have focused on tissue engineering of heart valves. The present study was designed to evaluate the implantation of a whole trileaflet tissue-engineered heart valve in the pulmonary position in a lamb model. METHODS AND RESULTS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold that was fabricated from a porous polyhydroxyalkanoate (pore size 180 to 240 microm; Tepha Inc). Vascular cells were harvested from ovine carotid arteries, expanded in vitro, and seeded onto our heart valve scaffold. With the use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and 2-cm segments of pulmonary artery were replaced by autologous cell-seeded heart valve constructs (n=4). One animal received an acellular valved conduit. No animal received any anticoagulation therapy. Animals were killed at 1, 5, 13, and 17 weeks. Explanted valves were examined histologically with scanning electron microscopy, biochemically, and biomechanically. All animals survived the procedure. The valves showed minimal regurgitation, and valve gradients were <20 mm Hg on echocardiography. The maximum gradient was 10 mm Hg with direct pressures. Macroscopically, the tissue-engineered constructs were covered with tissue, and there was no thrombus formation on any of the specimens. Scanning electron microscopy showed smooth flow surfaces during the follow-up period. Histological examination demonstrated laminated fibrous tissue with predominant glycosaminoglycans as extracellular matrix. 4-Hydroxyproline assays demonstrated an increase in collagen content as a percentage of native pulmonary artery (1 week 45.8%, 17 weeks 116%). DNA assays showed a comparable number of cells in all explanted samples. There was no tissue formation in the acellular control. CONCLUSIONS: Tissue-engineered heart valve scaffolds fabricated from polyhydroxyalkanoates can be used for implantation in the pulmonary position with an appropriate function for 120 days in lambs.

Functional living trileaflet heart valves grown in vitro.

BACKGROUND: Previous tissue engineering approaches to create heart valves have been limited by the structural immaturity and mechanical properties of the valve constructs. This study used an in vitro pulse duplicator system to provide a biomimetic environment during tissue formation to yield more mature implantable heart valves derived from autologous tissue. METHODS AND RESULTS: Trileaflet heart valves were fabricated from novel bioabsorbable polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing flow and pressure conditions. By use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and the valve constructs were implanted into 6 lambs (weight 19+/-2.8 kg). All animals had uneventful postoperative courses, and the valves were explanted at 1 day and at 4, 6, 8, 16, and 20 weeks. Echocardiography demonstrated mobile functioning leaflets without stenosis, thrombus, or aneurysm up to 20 weeks. Histology (16 and 20 weeks) showed uniform layered cuspal tissue with endothelium. Environmental scanning electron microscopy revealed a confluent smooth valvular surface. Mechanical properties were comparable to those of native tissue at 20 weeks. Complete degradation of the polymers occurred by 8 weeks. Extracellular matrix content (collagen, glycosaminoglycans, and elastin) and DNA content increased to levels of native tissue and higher at 20 weeks. CONCLUSIONS: This study demonstrates in vitro generation of implantable complete living heart valves based on a biomimetic flow culture system. These autologous tissue-engineered valves functioned up to 5 months and resembled normal heart valves in microstructure, mechanical properties, and extracellular matrix formation.

Tissue engineering of heart valves: in vitro experiences.

BACKGROUND: Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS: It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS: Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Prevention of glutaraldehyde-fixed bioprosthetic heart valve calcification by alcohol pretreatment: further mechanistic studies.

BACKGROUND AND AIM OF THE STUDY: Calcification is a major cause of failure of bioprosthetic heart valves derived from glutaraldehyde-crosslinked bovine pericardium or porcine aortic valve (PAV) cusps. Recently, we have shown that ethanol pretreatment of PAV cusps prevents calcification in animal models. METHODS AND RESULTS: In this study we showed that ethanol pretreatment was equally effective in preventing calcification of glutaraldehyde-crosslinked bovine pericardium (control Ca2+ = 121.16+/-7.49 microg/mg tissue; ethanol-pretreated Ca2+ = 2.95+/-0.78 microg/mg). Furthermore, other low-molecular weight alcohols such as methanol and isopropanol were also effective in mitigating calcification of PAV cusps. Storage of ethanol-pretreated cusps in glutaraldehyde before implantation allowed partial return of calcification, suggesting a role for ethanol-glutaraldehyde interactions in preventing calcification. However, when ethanol-pretreated cusps were stored in ethanolic glutaraldehyde up to one month, the anti-calcification effect of ethanol persisted. The conditions whereby PAV cusps were crosslinked in pure, non-aqueous, alcoholic glutaraldehyde solutions were also examined. The crosslinking was equivalent to the standard aqueous glutaraldehyde crosslinking as indicated by thermal denaturation temperatures (Td) obtained by differential scanning calorimetry (DSC) and resistance to collagenase digestion. However, these cusps had lower water content and showed a marked decrease in spin-lattice relaxation times (T1) obtained by solid-state proton nuclear magnetic resonance (NMR). Moreover, these cusps calcified heavily in the 21-day rat subdermal implants. Thus, alcohol treatment during glutaraldehyde crosslinking was not useful. CONCLUSION: Glutaraldehyde storage after ethanol pretreatment aggravates calcification; moreover, alcoholic-glutaraldehyde crosslinking solutions are not beneficial for anti-calcification. Ethanol pretreatment of glutaraldehyde-pretreated bovine pericardium prevents its calcification.

Neuroprotection mediated by changes in the endothelial actin cytoskeleton.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction.

Matrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.

A t(2;19)(p13;p13.2) in a giant invasive cardiac lipoma from a patient with multiple lipomatosis.

Cardiac lipomas occur infrequently but account for a significant portion of rare cardiac tumors. Common cutaneous lipomas have previously been associated with rearrangements of chromosome band 12q15, which often disrupt the high-mobility-group protein gene HMGIC. In this report, we describe the cytogenetic analysis of an unusual giant cardiac lipoma that exhibited myocardial invasion in a patient with a history of multiple lipomatosis (cutaneous lipoma, lipomatous gynecomastia, lipomatous hypertrophy of the interatrial septum, and dyslipidemia). Cytogenetic studies of cells derived from the cardiac lipoma demonstrated no abnormalities of chromosome 12, but did reveal a t(2;19)(p13;p13.2). A liposarcoma-derived oncogene (p115-RhoGEF) previously mapped to chromosome 19 and the low-density lipoprotein receptor gene (LDLR) previously mapped to chromosome band 19p13 were evaluated to determine whether they were disrupted by this translocation. Fluorescence in situ hybridization analyses assigned p115-RhoGEF to chromosome 19 in bands q13.2-q13.3 and mapped the LDLR to chromosome arm 19p in segment 13.2, but centromeric to the t(2;19) breakpoint. Thus, these genes are unlikely to be involved in the t(2;19)(p13;p13.2). Further studies of the regions of chromosomes 2 and 19 perturbed by the translocation in this unusual infiltrating cardiac lipoma will identify gene(s) that participate in adipocyte growth and differentiation and may provide insight into syndromes of multiple lipomatosis.