Elena Rabkin

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.

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.

Evidence for Increased Collagenolysis by Interstitial Collagenases-1 and -3 in Vulnerable Human Atheromatous Plaques

BACKGROUND Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. METHODS AND RESULTS Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1- and MMP-13-positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. CONCLUSIONS This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

An HMG-CoA Reductase Inhibitor, Cerivastatin, Suppresses Growth of Macrophages Expressing Matrix Metalloproteinases and Tissue Factor In Vivo and In Vitro

BackgroundUnstable 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 ResultsTo 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 · kg−1 · d−1; control group, n=9, saline 0.6 mL · kg−1 · 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 (≥0.01 &mgr;mol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages. ConclusionsThese 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.

Lipid Lowering by Diet Reduces Matrix Metalloproteinase Activity and Increases Collagen Content of Rabbit Atheroma A Potential Mechanism of Lesion Stabilization

BACKGROUND Proteolytic enzyme activity in lipid-rich atheroma may promote plaque rupture and precipitate acute coronary syndromes. This study tested the hypothesis that lipid lowering stabilizes plaques by reducing proteolytic activity. METHODS AND RESULTS We produced experimental atheroma in 33 rabbits by balloon injury and an atherogenic diet (0.3% cholesterol and 4.7% coconut oil) for 4 months. At that time, 15 rabbits were killed (baseline group). The remaining animals were divided into two groups: a hyperlipemic group continued to consume a cholesterol-enriched diet (0.05% to 0.2%) for 16 more months (n=5) and a lipid-lowering group consumed a purified chow diet with no added cholesterol or fat for 8 (n=3) or 16 months (n=10). Macrophage accumulation and interstitial collagenase (matrix metalloproteinase-1, MMP-1) expression in the lesion were measured by quantitative image analysis of standardized sections of immunostained aortas. Baseline lesions expressed high levels of MMP-1 and contained many macrophages. These features of plaque instability persisted in the hyperlipemic group. However, the lipid-lowering group showed progressive reduction in both macrophage content and MMP- 1 immunoreactivity with time. Aortic rings of the baseline and hyperlipemic groups elaborated gelatinolytic, caseinolytic, and elastinolytic activity attributable to MMP-2, MMP-3, or MMP-9, monitored by SDS-PAGE zymography. Proteolytic activity decreased markedly in the lipid-lowering group. Aortic content of interstitial collagen, determined by sirius red staining, increased in the lipid-lowering group compared with the baseline or continued hyperlipemic groups, indicating that lipid lowering reinforced the fibrous skeleton of the atheroma. CONCLUSIONS These results establish a mechanism by which lipid lowering may stabilize vulnerable plaques by reduced expression and activity of enzymes that degrade the arterial extracellular matrix and render atheroma less susceptible to disruption and thrombosis by favoring collagen accumulation in the fibrous cap.

Host bone-marrow cells are a source of donor intimal smooth- muscle–like cells in murine aortic transplant arteriopathy

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in β-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of β-galactosidase–expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle–like cells in GAD lesions can originate from circulating bone-marrow–derived precursors.

Statins Alter Smooth Muscle Cell Accumulation and Collagen Content in Established Atheroma of Watanabe Heritable Hyperlipidemic Rabbits

Background —Acute coronary syndromes often result from rupture of vulnerable plaques. The collagen content of plaques probably regulates their stability. This study tested whether HMG-CoA reductase inhibitors (statins) alter interstitial collagen gene expression or matrix metalloproteinase (MMP) levels in rabbit atheroma. Methods and Results —We administered equihypocholesterolemic doses of pravastatin (a hydrophilic statin, 50 mg · kg−1 · d−1, n=9), fluvastatin (a cell-permeant lipophilic statin, 20 mg · kg−1 · d−1, n=10), or placebo (n=10) to mature Watanabe heritable hyperlipidemic rabbits for 52 weeks. The fluvastatin group achieved a much higher peak plasma concentration (23.7 &mgr;mol/L) than did the pravastatin group (1.3 &mgr;mol/L) under these conditions. Immunohistochemistry revealed that MMP-1, MMP-3, and MMP-9 expression by macrophages in the intima was lower in both the pravastatin and fluvastatin groups than in the placebo group, whereas there was no difference in macrophage numbers. Numbers of intimal smooth muscle cells (SMCs) (identified by immunohistochemistry) and expression of type I procollagen mRNA (detected by in situ hybridization), however, were significantly higher in the pravastatin group than in the fluvastatin group. Treatment with pravastatin, but not fluvastatin, preserved interstitial collagen content in vivo (detected by picrosirius red polarization). In vitro, fluvastatin, but not pravastatin, decreased numbers of rabbit and human aortic SMCs without altering procollagen I mRNA expression. Conclusions —This study showed that statins can reduce MMP expression in atheroma and that cell-permeant statins can decrease SMC number and collagen gene expression in vivo.

From Stem Cells to Viable Autologous Semilunar Heart Valve

Background—An estimated 275 000 patients undergo heart valve replacement each year. However, existing solutions for valve replacement are complicated by the morbidity associated with lifelong anticoagulation of mechanical valves and the limited durability of bioprostheses. Recent advances in tissue engineering and our understanding of stem cell biology may provide a lifelong solution to these problems. Methods and Results—Mesenchymal stem cells were isolated from ovine bone marrow and characterized by their morphology and antigen expression through immunocytochemistry, flow cytometry, and capacity to differentiate into multiple cell lineages. A biodegradable scaffold was developed and characterized by its tensile strength and stiffness as a function of time in cell-conditioned medium. Autologous semilunar heart valves were then created in vitro using mesenchymal stem cells and the biodegradable scaffold and were implanted into the pulmonary position of sheep on cardiopulmonary bypass. The valves were evaluated by echocardiography at implantation and after 4 months in vivo. Valves were explanted at 4 and 8 months and examined by histology and immunohistochemistry. Valves displayed a maximum instantaneous gradient of 17.2±1.33 mm Hg, a mean gradient of 9.7±1.3 mm Hg, an effective orifice area of 1.35±0.17 cm2, and trivial or mild regurgitation at implantation. Gradients changed little over 4 months of follow-up. Histology showed disposition of extracellular matrix and distribution of cell phenotypes in the engineered valves reminiscent of that in native pulmonary valves. Conclusions—Stem-cell tissue-engineered heart valves can be created from mesenchymal stem cells in combination with a biodegradable scaffold and function satisfactorily in vivo for periods of >4 months. Furthermore, such valves undergo extensive remodeling in vivo to resemble native heart valves.

Lipid Lowering Reduces Oxidative Stress and Endothelial Cell Activation in Rabbit Atheroma

Background—Lipid lowering may reduce acute coronary events in patients in part by reducing vascular inflammation. Oxidative stress induces endothelial cell (EC) expression of vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemoattractant protein 1 (MCP-1) and reduces levels of atheroprotective NO, leading to monocyte recruitment and macrophage accumulation. This study tested the hypothesis that lipid lowering decreases oxidative stress and improves EC functions related to inflammatory cell accumulation. Methods and Results—Rabbits consumed an atherogenic diet for 4 months to produce atheroma, followed by a purified chow diet for 16 months. Atherosclerotic aortas from hypercholesterolemic rabbits produced high levels of reactive oxygen species. Oxidized LDL (oxLDL) accumulated in atheroma underlying ECs that overexpress VCAM-1. In contrast, few if any ECs in atheroma stained for endothelial NO synthase (eNOS). Lipid lowering reduced reactive oxygen species production, oxLDL accumulation, and plasma levels of anti-oxLDL IgG. After lipid lowering, VCAM-1 and MCP-1 expression decreased, eNOS expression increased, and ECs exhibited a more normal ultrastructure. Conclusions—These results establish that lipid lowering can reduce oxidative stress and EC activation in vivo. These mechanisms may contribute to improvement in endothelial function and plaque stabilization observed clinically.

Cardiovascular tissue engineering

Tissue engineering is a broad term describing a set of tools at the interface of the biomedical and engineering sciences that use living cells or attract endogenous cells to aid tissue formation or regeneration, and thereby produce therapeutic or diagnostic benefit. In the most frequent paradigm, cells are seeded on a scaffold composed of synthetic polymer or natural material (collagen or chemically treated tissue), a tissue is matured in vitro, and the construct is implanted in the appropriate anatomic location as a prosthesis [1–4]. A typical scaffold is a bioresorbable polymer in a porous configuration in the desired geometry for the engineered tissue, often modified to be adhesive for cells, in some cases selective for a specific cell population. Either application-specific differentiated or undifferentiated (stem) cells are used [5,6]. The first phase is the in vitro formation of a tissue construct by placing the chosen cells and scaffold in a metabolically and mechanically supportive environment with growth media (in a bioreactor), in which the cells proliferate and elaborate extracellular matrix (ECM). In the second phase, the construct is implanted in the appropriate anatomic location, where remodeling in vivo is intended to recapitulate the normal functional architecture of an organ or tissue. The key processes occurring during the in vitro and in vivo phases of tissue formation and maturation are (1) cell proliferation, sorting and differentiation; (2) ECM production and organization; (3) degradation of the scaffold; and (4) remodeling and potentially growth of the tissue. The general paradigm of tissue engineering is illustrated in Fig. 1. Since the cells of the implant can contact the surrounding tissues of the recipient, this approach is termed an ‘‘open-system.’’ In contrast, ‘‘closed-system’’ devices have therapeutic cells that are encapsulated or otherwise isolated from the recipient’s blood or tissues by semipermeable polymer membranes. This permits nutrients, wastes, drugs or hormones to pass yet keeps larger molecules such as the recipient’s antibodies or inflammatory/immune cells away, thus protecting the cells from degradation. Closed-system devices are used to deliver biologically active agents (such as drugs) to a restricted anatomic site (localized, controlled drug delivery) or to serve as extracorporeal cell-containing devices for renal, hepatic or pancreatic assist. Some tissue engineering applications use one of various ‘‘incomplete’’ paradigms, in which certain steps in the general paradigm are omitted (Fig. 2). For example, the cell-seeded scaffold model (see Fig. 2A) is exemplified by endothelial seeding of a synthetic or tissue derived vascular graft prior to implantation [7,8]. The cell transplant model (see Fig. 2B) is exemplified by one approach to myocardial tissue engineering in which injected cells (myocytic or with myocyte potential) differentiate and proliferate in or near damaged myocardium [9,10]. Investigators attempting to provide engineered heart valves and vascular grafts have utilized decellularized tissue scaffolds that attract endogenous cells (see Fig. 2C) to repopulate and remodel an altered tissue that preserves architectural and chemical information [11,12]. In vitro tissue ‘‘equivalents’’ (see Fig. 2D) may provide tools to understand normal physiological processes, study pathogenesis of disease and provide diagnostic and therapeutic tools (e.g., patterned cell cultures to probe cellcell and cell-matrix interactions and enhance cell-based drug discovery and target validation, vascular constructs to study contractile responses, and myocardial constructs for studying drug toxicities and responses to tissue injury) [13,13a,13b]. The innovative fabrication of materials and the development of sophisticated methods to repair or regenerate damaged or diseased tissues and to create entire organ replacements requires integration of a diverse array of basic scientific principles and enabling technologies. Thus, tissue engineering requires an understanding of relationships of structure to function in normal and pathological tissues