Use of autologous auricular chondrocytes for lining artificial surfaces: a feasibility study.

Background: Auricular elastic cartilage is a potential source of autologous cells for lining the luminal surfaces of cardiovascular prostheses. We tested this potential in vitro and in vivo using a left ventricular assist device (LVAD) and a calf model.

Methods: In vitro, auricular cartilage was harvested from the anesthetized ear of a calf, isolated, and cultured on tissue culture dishes.

Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition.

The goal of the current study was to evaluate matrix protein synthesis by cells cultured on materials that had been modified with cell adhesion ligands. We examined the effects of surface peptide density and of peptides with different affinities on the extracellular matrix production of smooth muscle cells, endothelial cells and fibroblasts. While initial adhesion was greatest on the higher density peptide surfaces, all cell types exhibited decreased matrix production on the more highly adhesive surfaces.

A genetically engineered, nonthrombogenic cellular lining for LVADs: in vitro preconditioning before in vivo implantation.

Because of the clinical success of left ventricular assist devices (LVADs) used for short-term "bridge to transplant" and the limited availability of donor organs, heart assist devices are being considered for long-term implantation as an alternative to heart transplantation. In an effort to improve biocompatibility, our laboratory has developed a nonthrombogenic cellular lining from genetically engineered smooth muscle cells (GE-SMC) for the Thermocardiosystems Heartmate LVAD. Smooth muscle cells have been transduced with the gene for endothelial nitric oxide synthase (NOS III) and produce NO at concentrations that reduce platelet deposition and smooth muscle cell proliferation when tested in vitro.

Nonthrombogenic, adhesive cellular lining for left ventricular assist devices.

Background: The textured, blood-contacting surfaces of the Thermocardiosystems HeartMate left ventricular assist device (LVAD) promote the passivation of the biomaterial caused by the accumulation of an integral coagulum. Commonly, acute, postimplantation thrombocytopenia causes significant bleeding, requiring surgery or blood transfusions. Chronic complications include thromboembolic microevents that can affect central nervous system function.

Stimulation of growth and migration of vascular endothelial cells by vascular endothelial growth factor transduced smooth muscle cells in co-culture.

Vascular endothelial growth factor (VEGF) is a secreted mitogen with high specificity toward endothelial cells. Expression of VEGF by smooth muscle cells in vivo may be an important stimulus for the regrowth of the endothelium after damage caused by interventions such as angioplasty. The levels of VEGF secreted by cultured smooth muscle cells minimally stimulated growth of endothelial cells in co-culture.

Liposomal induction of NO synthase expression in cultured vascular smooth muscle cells.

Transfection of bovine smooth muscle cells with plasmid constructs containing the full coding sequence for endothelial NO synthase (NOS3) using liposome mediated gene transfer gave rise to cells that produced high levels of NO. Western analysis indicated that transfected cells were indeed expressing NOS3 protein, but in addition expression of inducible NO synthase (NOS2) was detected. The latter accounted for the high levels of NO produced by transfectants.

Genetically engineered smooth muscle cells as linings to improve the biocompatibility of cardiovascular prostheses.

Background: The seeding of the blood-contacting surfaces of cardiovascular prostheses with autologous endothelial cells to improve their biocompatibility has had little success. In most instances, cells have sloughed off under flow conditions. The performance of left ventricular assist devices (LVADs) designed to stabilize patients awaiting donor hearts for transplantation has been remarkably good.

Regulation of interleukin-1-beta-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells by hemostatic proteins.

Experiments were performed to examine the mechanism by which specific hemostatic proteins regulate the release of nitric oxide (NO) from interleukin-1 beta (IL-1beta) stimulated cultured rate aortic smooth muscle cells. Treatment of smooth muscle cells with IL-Beta stimulated inducible nitric oxide synthase (iNOS) mRNA expression, which preceded the release of NO (as measured by the accumulation of nitrite in the culture media). The cytokine-stimulated production of nitrite was blocked by the protein synthesis inhibitor cycloheximide, the transcriptional inhibitor actinomycin D, and the competitive inhibitor of NOS nitro-L-arginine.

Endogenous and exogenous nitric oxide protect against intracoronary thrombosis and reocclusion after thrombolysis.

Background: Nitric oxide (NO), an endothelium-derived relaxing factor, plays an important role in regulating platelet activation. We evaluated the effect of NO in a canine model of intracoronary thrombosis, thrombolysis, and reocclusion.

Methods And Results: Before thrombosis was induced, 34 anesthetized dogs were treated with a continuous intracoronary infusion of saline (n = 8); NG-nitro-L-arginine (L-NNA, n = 8), an inhibitor of NO synthetase; L-arginine (n = 7), the precursor for NO; or sodium nitroprusside (SNP, n = 11), an NO donor.

Pathophysiology of smooth muscle in hypertension.

Structural changes of the arteries in hypertension are determined by the unique genetics of the animals and by various growth promoters and growth inhibitors. Vascular smooth muscle cell growth promoting factors include fibroblast growth factor, platelet-derived growth factor, and vasoactive peptides such as norepinephrine, angiotensin II, and endothelin. Endothelial cells secrete three types of growth inhibiting factors.

The predominant form of fibroblast growth factor receptor expressed by proliferating human arterial smooth muscle cells in culture is type I.

Fibroblast growth factors (FGF) and their specific receptors (FGFR) have diverse roles, including induction of proliferation in smooth muscle cells which contributes to restenosis after coronary artery balloon angioplasty. The relative levels of expression of the four major types of FGFR were studied in 13 different human arterial smooth muscle cell isolates. Cell lines were established by the explant technique from intima/media tissue samples obtained from patients undergoing either coronary artery bypass surgery or cardiac transplantation procedures.

DL-propranolol augments production of NO induced by cytokines in cultured aortic smooth muscle of the rat.

The effect of DL-propranolol on the production of nitric oxide (NO.) by cultured arterial smooth muscle cells from normotensive (WKY) and spontaneously hypertensive rats (SHR) was studied before and after stimulation by lipopolysaccharide or interleukin-1 beta. The influence of L-arginine and NG-nitro-L-arginine on these events was also studied.

Simultaneous activation of adenylyl cyclase and protein kinase C induces production of nitric oxide by vascular smooth muscle cells.

Rat aortic smooth muscle cells produced large quantities of nitric oxide (NO) after exposure to interleukin-1 beta, and this was depressed in the presence of the protein kinase C inhibitor bisindolylmaleimide. Intracellular cAMP levels were elevated mildly in cytokine-treated smooth muscle cells, and the presence of forskolin enhanced both the cAMP levels and NO production. Inhibition of GTP:cyclohydrolase I by 2,4-diamino-6-hydroxypyrimidine attenuated NO production by interleukin-1 beta-treated cells.

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells.

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels.

Regulation of smooth muscle cell growth by endothelium-derived factors.

The endothelium is a source of molecules that either stimulate or inhibit the proliferation of the underlying smooth muscle cells. In the normal, healthy vessel wall the smooth muscle cells are quiescent, but they proliferate when damage to the endothelium occurs. The implication of such observations is that although the endothelium provides a source of growth factors, their stimulatory activity on smooth muscle cells is countered by endothelium-derived growth inhibitors.

The endothelium in health and disease.

The early observations of an apparent anomalous action of acetylcholine on the regulation of vascular tone in vivo and in vitro were found to be a reflection of the intactness of the endothelium in vivo. An intact endothelium mediates relaxation of smooth muscle in response to acetylcholine, whereas endothelium-denuded blood vessels exposed to this agonist often exhibit vasoconstriction. The vasodilation is mediated by the actions of the endothelium-derived relaxing factors nitric oxide and prostacyclin.

Growth factor regulation of interleukin-1 beta-induced nitric oxide synthase and GTP: cyclohydrolase expression in cultured smooth muscle cells.

Induction of NO synthase expression by interleukin-1 beta in cultured vascular smooth muscle cells from rat aortas was accompanied by simultaneous induction of GTP: cyclohydrolase I. This enzyme regulates the de novo synthesis pathway for tetrahydrobiopterin, an essential cofactor for the catalytic conversion of L-arginine to L-citrulline and NO by inducible NO synthase. Inhibition of GTP: cyclohydrolase attenuated NO production by interleukin-1 beta-stimulated smooth muscle cells.

Enhanced production of nitric oxide in aortae from spontaneously hypertensive rats by interleukin-1 beta.

Cultured aortic smooth muscle cells from spontaneously hypertensive rats produce more nitrite than cells from Wistar-Kyoto rats in response to interleukin-1 beta. Therefore, the effect of interleukin-1 beta-induced nitric oxide production was compared on the contractility of aortic smooth muscle from spontaneously hypertensive and Wistar-Kyoto rats. Under control conditions, there was no difference in the response of aortic rings (without endothelium) to phenylephrine between both strains.

Effects of peptide vasoconstrictors on vessel structure.

The peptide vasoconstrictors angiotensin II and endothelin-1, originally described as being derived exclusively from the plasma renin-angiotensin system and vascular endothelium, respectively, have been demonstrated to be produced independently of these sources. Local tissue angiotensin-generating systems are well documented and endothelin production has been demonstrated for a variety of nonendothelial cells, including vascular smooth muscle cells. There is increasing evidence that these locally produced vasoconstrictor peptides may contribute to blood vessel homeostasis, as well as the development of vascular pathologic conditions.

Thrombin inhibits induction of nitric oxide synthase in vascular smooth muscle cells.

Experiments were designed to examine whether thrombin affects the production of nitric oxide-like factor(s) evoked by interleukin-1 beta (IL-1 beta) in cultured smooth muscle cells from the rat aorta. IL-1 beta stimulated the release of nitrite (a stable oxidation product of nitric oxide) from cultured smooth muscle cells. Thrombin inhibited in a concentration-dependent manner the release of nitrite caused by IL-1 beta.

Platelet-derived growth factor suppresses and fibroblast growth factor enhances cytokine-induced production of nitric oxide by cultured smooth muscle cells. Effects on cell proliferation.

Stimulation of thymidine incorporation by basic fibroblast growth factor or epidermal growth factor treatment of cultured quiescent smooth muscle cells (rat and human) was attenuated by the concomitant treatment with interleukin-1 beta in the presence of indomethacin. Platelet-derived growth factor-AB and -BB-induced thymidine incorporation was not inhibited by the presence of the cytokine under similar experimental conditions. Elevation of nitrite levels in the conditioned medium of cultures exposed to interleukin-1 beta correlated with the inhibition of thymidine incorporation.

Expression of tenascin by vascular smooth muscle cells. Alterations in hypertensive rats and stimulation by angiotensin II.

The extracellular matrix glycoprotein tenascin is associated with remodeling events in many embryonic and pathologic tissues. The expression of tenascin has been investigated by immunohistochemistry in blood vessels of Wistar-Kyoto (normotensive) and spontaneously hypertensive rats. Weak tenascin staining was present throughout the tunica media of large and small arteries from normotensive animals; strong staining was only detectable at branching sites.