Patricia M. Taylor

The cardiac valve interstitial cell

Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) alpha-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function.

Induction of vascular adhesion molecules during rejection of human cardiac allografts.

Adhesion of leukocytes to vascular endothelium is a necessary step leading to the migration of cells into underlying tissues. Vascular adhesion molecules regulate this process and may play an important role in graft rejection. Immunocytochemical studies have been used to investigate the expression of vascular adhesion molecules (ICAM-1, PEC AM, VCAM-1, and ELAM-1) in normal donor heart (n = 15) and myocardial biopsies from heart transplant patients with acute rejection (n = 15). Sections were also stained with antibodies against endothelium, leukocytes, MHC antigens, and markers of cell activation. In donor heart EN4, vWF, ICAM-1, PECAM, MHC class I—and, to a lesser extent, VCAM-1 and DR antigen—are expressed on arterioles and venules, whereas ELAM-1 and Pal-E are restricted to venules. Expression of Pal-E, VCAM-1, ICAM-1, and DR antigen was increased during rejection. Capillary endothelium normally expresses EN4, ICAM-1, PECAM, MHC class I, and DR antigen but little, if any, VCAM-1 or ELAM-1. During rejection, however, there is an increased expression of all adhesion molecules. This is paralleled by an increased expression of vWF by capillary endothelium. In addition, ICAM-1 like MHC class I antigen is induced on the myocardial membrane and intercalating discs. Endocardium from donor heart expresses EN4, vWF, PECAM, MHC class I, and sometimes Pal-E and ICAM-1, but very little VCAM-1, ELAM-1 or DR antigen. There is an increased expression of Pal-E, ICAM-1, VCAM-1, and DR antigen on endocardium from rejecting heart biopsies. Proliferating Ki-67+ cells and activated T cells expressing the receptor for IL-2 were also found in biopsies during rejection episodes.

Molecular and functional characteristics of heart-valve interstitial cells

The cells that reside within valve cusps play an integral role in the durability and function of heart valves. There are principally two types of cells found in cusp tissue: the endothelial cells that cover the surface of the cusps and the interstitial cells (ICs) that form a network within the extracellular matrix (ECM) within the body of the cusp. Both cell types exhibit unique functions that are unlike those of other endothelial and ICs found throughout the body. The valve ICs express a complex pattern of cell-surface, cytoskeletal and muscle proteins. They are able to bind to, and communicate with, each other and the ECM. The endothelial cells on the outflow and inflow surfaces of the valve differ from one another. Their individual characteristics and functions reflect the fact that they are exposed to separate patterns of flow and pressure. In addition to providing a structural role in the valve, it is now known that the biological function of valve cells is important in maintaining the integrity of the cusps and the optimum function of the valve. In response to inappropriate stimuli, valve interstitial and endothelial cells may also participate in processes that lead to valve degeneration and calcification. Understanding the complex biology of valve interstitial and endothelial cells is an important requirement in elucidating the mechanisms that regulate valve function in health and disease, as well as setting a benchmark for the function of cells that may be used to tissue engineer a heart valve.

Expression of MHC antigens in normal human lungs and transplanted lungs with obliterative bronchiolitis.

Immunocytochemical techniques have been used to investigate the expression of common determinants of class I, common determinants of class II and DR, DP, and DQ antigens on frozen sections from twenty normal donor lungs, three lungs resected due to carcinoma and nine lungs removed from heart/lung recipients who were undergoing retransplant due to obliterative bronchiolitis. In all lungs, alveolar macrophages expressed common determinants of class I and class II, as well as DR, DP and DQ antigens. In normal lung, class I was expressed on all vascular endothelium, all tracheal epithelium and most, but not all, bronchiolar epithelium. Class II was always expressed on tracheal epithelium, but expression on bronchiolar epithelium and vascular endothelium was variable and sometimes absent. In lungs from transplant patients with severe obliterative bronchiolitis, vascular endothelium, in addition to expressing class I, now consistently expressed class II. Epithelium from trachea, bronchioles, and alveoli also consistently expressed class I and class II. To conclude, there is enhanced expression of class II antigens on endothelial and epithelial cells from lungs with obliterative bronchiolitis.

Endothelium-Dependent Regulation of the Mechanical Properties of Aortic Valve Cusps

OBJECTIVES The aim of this study was to evaluate the role of valve endothelium in regulating the mechanical properties of aortic valve cusps. BACKGROUND Mechanical properties of valve cusps are key to their function and durability; however, little is known about the regulation of valve biomechanics. METHODS Mechanical properties of porcine aortic valve leaflets were evaluated in response to serotonin (5-hydroxytryptamine [5-HT]), with and without N-nitro-L-arginine-methyl-ester (L-NAME) or endothelial denudation, and endothelin (ET)-1, with and without cytochalasin-B. RESULTS Under physiological loading conditions, 5-HT induced a decrease in the areal stiffness of the cusp (-25.0 +/- 4.0%; p < 0.01 vs. control), which was reversed by L-NAME or endothelial denudation (+17.5 +/- 5.3%, p = 0.07, and +14.7 +/- 1.8%, p < 0.05 vs. control, respectively). ET-1 caused an increase in stiffness (+34.4 +/- 13.8%; p < 0.05 vs. control), but not in the presence of cytochalasin-B (p = 0.29 vs. control). Changes in cusp stiffness were accompanied by aortic cusp relaxations to 5-HT (-0.29% +/- 0.1% change in load per 10-fold increase in 5-HT concentration; p = 0.03), which were reversed by endothelial denudation (+0.29 +/- 0.06% change in load per 10-fold increase in 5-HT concentration; p = 0.02) and by L-NAME (p < 0.05). Valve cusps contracted in response to ET-1 (+0.29 +/- 0.08% change in load per 10-fold increase in ET-1 concentration; p = 0.02), which was inhibited by cytochalasin-B. CONCLUSIONS These data highlight the role of the endothelium in regulating the mechanical properties of aortic valve cusps and underline the importance of valve cellular integrity for optimal valve function.

Expression of adhesion molecules by Lp(a): a potential novel mechanism for its atherogenicity

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor‐mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM‐1) and E‐selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor‐activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of α2‐macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM‐1 and E‐selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor‐related protein nor cell‐surface proteoglycans are involved in Lp(a)‐induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM‐1 and E‐selectin were not significantly decreased by the addition of glu‐plasminogen, the lysine analog ε‐aminocaproic acid, or by trans‐4‐(aminomethyl)‐cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r‐apo(a)] competed with Lp(a) and attenuated the expression of VCAM‐1 and E‐selectin. In summary, we have identified a calcium‐dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM‐1 and E‐selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell‐activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.—Allen, S., Khan, S., Tam, S.‐P., Koschinsky, M., Taylor, P., Yacoub, M. Expression of adhesion molecules by Lp(a): a potential novel mechanism for its atherogenicity. FASEB J. 12, 1765–1776 (1998)

Extracellular matrix production by adipose-derived stem cells: implications for heart valve tissue engineering.

A key challenge in tissue engineering a heart valve is to reproduce the major tissue structures responsible for native valve function. Here we evaluated human adipose-derived stem cells (ADSCs) as a source of cells for heart valve tissue engineering investigating their ability to synthesize and process collagen and elastin. ADSCs were compared with human bone marrow mesenchymal stem cells (BmMSCs) and human aortic valve interstitial cells (hVICs). ADSCs and BmMSCs were stretched at 14% for 3 days and collagen synthesis determined by [(3)H]-proline incorporation. Collagen and elastin crosslinking was assessed by measuring pyridinoline and desmosine respectively, using liquid chromatography/mass spectrometry. Three-dimensional culture was obtained by seeding cells onto bovine collagen type I scaffolds for 2-20 days. Expression of matrix proteins and processing enzymes was assessed by Real Time-PCR, immunofluorescence and transmission electron microscopy. Stretch increased the incorporation of [(3)H]-proline in ADSCs and BmMSCs, however only ADSCs and hVICs upregulated COL3A1 gene. ADSCs produced collagen and elastin crosslinks. ADSCs uniformly populated collagen scaffolds after 2 days, and fibrillar-like collagen was detected after 20 days. ADSCs sense mechanical stimulation and produce and process collagen and elastin. These novel findings have important implications for the use of these cells in tissue engineering.

Transforming Growth Factor Beta 1 and Hyaluronan Oligomers Synergistically Enhance Elastin Matrix Regeneration by Vascular Smooth Muscle Cells

Elastin is a vital structural and regulatory matrix protein that plays an important role in conferring elasticity to blood vessel wall. Previous tissue engineering approaches to regenerate elastin in situ or within tissue engineering constructs are curtailed by innate poor elastin synthesis potential by adult vascular smooth muscle cells (SMCs). Currently, we seek to develop cellular cues to enhance tropoelastin synthesis and improve elastin matrix yield, stability, and ultrastructure. Our earlier studies attest to the elastogenic utility of hyaluronan (HA)-based cellular cues, though their effects are fragment size dependent and dose dependent, with HA oligomers deemed most elastogenic. We presently show transforming growth factor beta 1 (TGF-beta1) and HA oligomers, when provided concurrently, to synergistically and dramatically improve elastin matrix regeneration by adult vascular SMCs. Together, these cues suppress SMC proliferation, enhance synthesis of tropoelastin (8-fold) and matrix elastin protein (5.5-fold), and also improve matrix elastin yield (45% of total elastin vs. 10% for nonadditive controls), possibly by more efficient recruitment of tropoelastin for crosslinking. The density of desmosine crosslinks within the elastin matrix was itself attenuated, although the cues together modestly increased production and activity of the elastin crosslinking enzyme, lysyl oxidase. TGF-beta1 and HA oligomers together induced much greater assembly of mature elastin fibers than they did separately, and did not induce matrix calcification. The present outcomes might be great utility to therapeutic regeneration of elastin matrix networks in situ within elastin-compromised vessels, and within tissue-engineered vascular graft replacements.

Regulation of ecto-5′-nucleotidase by TNF-α in human endothelial cells

Ecto-5′-nucleotidase (E5′N, CD73) is key enzyme responsible for formation of anti-inflammatory and immunosuppressive adenosine from extracellular nucleotides as well as an important surface molecule involved in cellular signalling. In this study we provide evidence that the pro-inflammatory cytokine, tumour necrosis factor-α (TNF-α) may reduce the capacity of human endothelial cells to produce adenosine by a decrease in surface expression and in the activity of E5′N. Human umbilical vein endothelial cells incubated for 24 h with TNF-α lost 54% of the activity of E5′N while activities of the other enzymes involved in adenosine metabolism remained unaffected. Immunofluorescence staining with anti-E5′N (1E9) following exposure to TNF-α, showed reduced numbers of positive cells. TNF-α induced down-regulation of E5′N was prevented by addition of the PLC inhibitor neomycin, but not by inhibitors of MAPK-like pathways (MEK and p38). Therefore, we conclude that TNF-α through activation of endogenous PLC leads to cleavage of the GPI-linkage of E5′N resulting in loss of E5′N from the extracellular surface. This change may lead to decrease in formation of adenosine and could be an important mechanism of endothelial activation during inflammation.

Biological matrices and bionanotechnology.

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.