Robert M. Nerem

Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.

The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.

Dynamic Mechanical Conditioning of Collagen-Gel Blood Vessel Constructs Induces Remodeling In Vitro

AbstractDynamic mechanical conditioning is investigated as a means of improving the mechanical properties of tissue-engineered blood vessel constructs composed of living cells embedded in a collagen-gel scaffold. This approach attempts to elicit a unique response from the embedded cells so as to reorganize their surrounding matrix, thus improving the overall mechanical stability of the constructs. Mechanical conditioning, in the form of cyclic strain, was applied to the tubular constructs at a frequency of 1 Hz for 4 and 8 days. The response to conditioning thus evinced involved increased contraction and mechanical strength, as compared to statically cultured controls. Significant increases in ultimate stress and material modulus were seen over an 8 day culture period. Accompanying morphological changes showed increased circumferential orientation in response to the cyclic stimulus. We conclude that dynamic mechanical conditioning during tissue culture leads to an improvement in the properties of tissue-engineered blood vessel constructs in terms of mechanical strength and histological organization. This concept, in conjunction with a proper biochemical environment, could present a better model for engineering vascular constructs.

Oscillatory Shear Stress Stimulates Adhesion Molecule Expression in Cultured Human Endothelium

Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by approximately 70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.

Phosphorylation of Endothelial Nitric Oxide Synthase in Response to Fluid Shear Stress

Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+]i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm2) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+]i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+]i with a novel flow-step protocol. [Ca2+]i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+]i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.

Shear Stress Modulates Expression of Cu/Zn Superoxide Dismutase in Human Aortic Endothelial Cells

A major determinant of the level of cellular superoxide anion (O2-.) is the dismutation of O2-. to hydrogen peroxide by the enzyme superoxide dismutase (SOD). Three forms of SOD exist, but in endothelial cells, the major form outside of the mitochondria is the cytosolic copper/zinc-containing superoxide dismutase (Cu/Zn SOD). Since fluid shear stress is an important determinant of the function and structure of endothelial cells in vivo, we examined the effect of laminar shear stress on the expression of Cu/Zn SOD in cultured human aortic endothelial cells. Laminar shear stress of 0.6 to 15 dyne/cm2 increased Cu/Zn SOD mRNA in a time- and dose-dependent manner in human aortic endothelial cells. Shear stress also increased both Cu/Zn SOD protein content and the enzyme activity. Nuclear runon assays showed that nuclei from human aortic endothelial cells exposed to laminar shear stress had a 1.6-fold greater transcriptional activity of the Cu/Zn SOD gene compared with cells not exposed to shear, indicating that an increase in Cu/Zn SOD mRNA induced by laminar shear stress is at least in part mediated by increased transcription. In contrast, shear stress had no effect on Cu/Zn SOD mRNA levels in human aortic smooth muscle cells. These findings show that physiological levels of shear stress increase expression of Cu/Zn SOD in the endothelium. This adaptation to shear stress might augment the effect of locally produced NO. and thereby promote the antiatherogenic and anti-inflammatory properties of the endothelial cell.

Tissue engineering: from biology to biological substitutes.

Tissue engineering is an emerging multidisciplinary and interdisciplinary field involving the development of bioartificial implants and/or the fostering of tissue remodeling with the purpose of repairing or enhancing tissue or organ function. Bioartificial constructs generally consist of cells and biomaterials, so tissue engineering draws from both cell and biomaterials science and technology. Successful applications require a thorough understanding of the environment experienced by cells in normal tissues and by cells in bioartificial devices before and after implantation. This paper reviews these topics, as well as the current status and future possibilities in the development of different bioartificial constructs, including bioartificial skin, cardiovascular implants, bioartificial pancreas, and encapsulated secretory cells. Issues that need to be addressed in the future are also discussed. These include, but are not limited to, the development of new cell lines and biomaterials, the evaluation of the optimal construct architecture, and the reproducible manufacture and preservation of bioartificial devices until ready for use.

Effects of pulsatile flow on cultured vascular endothelial cell morphology.

Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS)

Unique Morphology and Focal Adhesion Development of Valvular Endothelial Cells in Static and Fluid Flow Environments

Background—The influence of mechanical forces on cell function has been well documented for many different cell types. Endothelial cells native to the aortic valve may play an important role in mediating tissue responses to the complex fluid environment, and may therefore respond to fluid flow in a different manner than more characterized vascular endothelial cells. Methods and Results—Porcine endothelial cells of aortic and aortic valvular origin were subjected to 20 dynes/cm2 steady laminar shear stress for up to 48 hours, with static cultures serving as controls. The aortic valve endothelial cells were observed to align perpendicular to flow, in direct contrast to the aortic endothelial cells, which aligned parallel to flow. Focal adhesion complexes reorganized prominently at the ends of the long axis of aligned cells. Valvular endothelial cell alignment was dependent on Rho-kinase signaling, whereas vascular endothelial cell alignment was dependent on both Rho-kinase and phosphatidylinositol 3-kinase signal pathways. Conclusions—These differences in response to mechanical forces suggest a unique phenotype of valvular endothelial cells not mimicked by vascular endothelial cells, and could have implications for cardiovascular cell biology and cell-source considerations for tissue-engineered valvular substitutes.

Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation.

AbstractBiochemical stimulation was applied in combination with cyclic mechanical strain to engineered vascular constructs made of isolated smooth muscle cells in a three-dimensional (3D) collagen type I matrix. Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β) were added exogenously to the medium used to culture the constructs. Mechanical stimulation was applied using a bioreactor system that imparted a 10% circumferential strain at a frequency of 1 Hz. The parameters studied were gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA). Mechanical stimulation caused a characteristic increase in gel compaction and cell proliferation, relative to statically cultured controls. Stimulation with PDGF increased cell proliferation and decreased SMA expression in 3D gels, but inhibited the effects of mechanical stimulation and produced a more open matrix structure. TGF-β strongly inhibited cell proliferation and increased SMA expression, especially in the presence of mechanical strain, and resulted in a dense matrix. These results show that cell phenotype can be modulated in engineered blood vessels by applying selected combinations of biochemical and mechanical stimuli, and suggest that such control over cell function can be used to tailor the properties of engineered tissues.

The Study of the Influence of Flow on Vascular Endothelial Biology

It is now recognized that the mechanical environment of a cell has an influence on its structure and function. For the vascular endothelial cell that resides at the interface of the flowing blood and the underlying vessel wall, there is mounting evidence of the importance of flow and the associated wall shear stress in the regulation of endothelial biology. Not only is it a sensitive regulator of endothelial structure and function, but different flow environments will influence endothelial cell biology differently. Furthermore, there may be an interaction between the chemical environment of a cell and its mechanical environment. This is illustrated by the inhibition by steady laminar shear stress of the cytokine induction of VCAM-1. Results also are presented in which flow studies have been conducted using a co-culture model of the vessel wall. These experiments provide evidence of a quiescent endothelium; however, much more needs to be done to engineer the cell culture environment to make it more physiologic.