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. Similarly, when different peptides were evaluated, matrix production was the lowest on the most adhesive surface and highest on the least adhesive surface. These results suggest that extracellular matrix synthesis may be regulated, to some extent, by signal transduction initiated by adhesion events. This may pose limitations for use of bioactive materials as tissue engineering scaffolds, as matrix production is an important aspect of tissue formation. However, it may be possible to increase matrix production on highly adhesive surfaces using exogenous factors. TGF-beta was shown to increase matrix production by both smooth muscle cells and endothelial cells.

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. In this investigation, the adhesive capabilities of GE-SMC linings were examined. An in vitro circulatory loop was designed to expose cell lined LVADs to in vivo operating conditions. Cumulative cell loss from cell lined LVADs was less than 10% after 24 hours of flow. Using a protocol for "preconditioning" the cell lining within the mock circulatory loop, the first implantation of an LVAD containing a genetically engineered SMC lining was successfully implemented in a bovine model. Results from this 24 hour study indicate that the flow-conditioned cellular lining remained intact with no evidence of thromboembolization and only minimal changes in coagulation studies.

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. Pumps, explanted during donor organ transplantation, are often found to have an extensive cellular panus associated with the blood-contacting surfaces of the device. This natural cellular lining suggests a possible strategy for improving the blood biocompatibility of the HeartMate. Therefore, seeding of LVADs with cells genetically engineered to enhance their antithrombotic properties before implantation was investigated as a means to improve biocompatibility for long-term use. METHODS AND RESULTS: Bovine vascular smooth muscle cells genetically engineered to produce nitric oxide were seeded on LVAD biomaterials and exposed to elevated shear stresses to determine cell-adhesive capabilities. Comparative studies were performed with vascular endothelial cells isolated from the same vessel. To assess the thrombogenic potential of the genetically engineered smooth muscle cells, monolayers were exposed to whole blood in parallel plate flow chambers and were platelet-adhesion quantified. This procedure used scanning electron microscopy and computer image-capture software. Endothelial cell monolayers and mock-transduced smooth muscle cells were assayed in a comparative manner. LVADs were seeded with genetically engineered smooth muscle cells and maintained under cell culture conditions for 96 hours. Thereafter, seeded LVADs were incorporated into in vitro flow loops. Cell retention within the pump was determined by sampling the effluent culture medium downstream of the pump and cell counting in a Coulter counter. After 18 hours of in vitro flow, a seeded pump was implanted into the abdominal cavity of a calf and anastomosed to the apex of the heart and to the descending aorta. More genetically engineered smooth muscle cells were retained on the surface of LVAD biomaterials when they were subjected to shear stresses up to 75 dyne/cm than endothelial cells assayed in the identical manner. Adherence of platelets to the surface of smooth muscle cells was significantly reduced after their transduction with nitric oxide synthase with GTP cyclohydrolase genes. Platelet deposition on the genetically modified myocyte layers was similar to that associated with endothelial cell layers. Cell loss from cell-seeded LVADs incorporated into in vitro flow loops remained < 5% of the total cell number seeded regardless of the duration of flow. CONCLUSIONS: LVADs seeded with smooth muscle cells, transduced with the genes to optimize nitric oxide production, adhered well to the pump surface under in vitro and in vivo flow conditions.

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. Full length cDNA for the 165 amino acid residue, bovine VEGF (VEGF165), was isolated from calf liver total RNA by reverse transcriptase polymerase chain reaction (RT-PCR) techniques, and used to generate plasmid constructs for transfection. Bovine aortic smooth muscle cells (BSMC), stably transfected with VEGF165 plasmid DNA, secreted mitogen into conditioned culture medium at levels that are physiologically relevant (2-4 ng/ml). Transformed BSMC stimulated growth of bovine aortic endothelial cells (BAEC) in co-culture, to a significantly greater extent than mock transfected BSMC. Migration of BAEC was also enhanced by the presence of VEGF transduced BSMC. These data suggest that smooth muscle cells, genetically engineered to produce VEGF, may provide biologic linings in cardiovascular prostheses that could promote the growth of endogenous endothelial cells.

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. Treatment of bovine or rat smooth muscle cells or 3T3 fibroblasts with only liposome preparations resulted in the induction of NOS2 expression and NO production. All liposomal reagents were shown to be endotoxin free. Direct induction of gene expression by liposomes alone suggests caution in interpretation of data for which gene transfer is mediated by liposomal preparations.

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. After prolonged implantation, pump surfaces become covered with a pannus of smooth muscle-like cells (myofibroblasts). Occasional islands of endothelial cells have been identified on top of such cell layers. Therefore, in an attempt to accelerate the beneficial conditioning and improve biomaterial-blood compatibility of LVAD internal surfaces, their seeding with autologous, genetically engineered smooth muscle cells (SMCs) was investigated. METHODS AND RESULTS: Since routine testing of the Thermocardiosystems HeartMate LVAD is carried out in calves, SMCs were isolated from calves, propagated in culture, and transduced with NO synthase genes to yield stable production of NO. Previous studies had demonstrated that SMCs attached strongly to the biomaterials that compose the internal surfaces of LVADs. Transduction of NO synthase gene expression in the SMCs was achieved by electroporation and antibiotic (G418) selection. Inhibition of smooth muscle cell proliferation by NO has been documented, and the same molecule has been shown to inhibit platelet adhesion to cell surfaces. Cells transduced with NO synthase expressed enzyme protein at consistently high levels for several passages in culture; however, NO production was dependent on the supplementation of culture medium with a source of tetrahydrobiopterin (sepiapterin). Under such conditions, transduced cells were growth-inhibited compared with mock-transfected controls. Induction of GTP cyclohydrolase (the rate-limiting enzyme for the production of tetrahydrobiopterin) expression also resulted in NO production by NO synthase-transduced cells. CONCLUSIONS: Preliminary studies have shown that SMCs form strong attachments to the surface materials of LVADs and that their proliferation rates could be controlled after transformation with NO synthase under conditions that support production of NO. Therefore, genetically engineered SMCs may provide an improved blood biomaterial interface for cardiovascular prostheses.

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. However, only actinomycin D inhibited IL-1beta-stimulated iNOS mRNA expression, Treatment of smooth muscle cells with IL-1beta in the presence of platelet derived growth factor or thrombin resulted in the inhibition of cytokine-stimulated expression of iNOS mRNA and NO release. The inhibitory effect of thrombin was reversed by hirudin and was mimicked by a 14 amino acid thrombin receptor activating peptide. In contract, the concomitant exposure of smooth muscle cells to IL-1beta-and plasmin resulted resulted in the potentiation of both IL-1beta-stimulated iNOS expression and NO generation. Finally, treatment of smooth muscle cells with IL-1beta in the presence of the hemostatic proteins did not affect the half-life of iNOS mRNA. These results demonstrate that specific protein components of the hemostatic system regulate IL- 1beta-stimulated iNOS mRNA expression in vascular smooth muscle cells. The capacity of hemostatic proteins to modulate the induction of vascular iNOS activity may play an important role in governing the release of NO and regulating thrombogenesis in vivo.

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. Ten minutes after the infusion was begun, an electric current of 150 microA was applied to the endothelium of coronary arteries to induce thrombosis. Occlusive thrombi developed in all dogs in the saline group (38 +/- 4 minutes) and the L-NNA group (30 +/- 6 minutes), in 6 of 7 dogs in the L-arginine group (81 +/- 18 minutes), and in 6 of 11 dogs in the SNP group (102 +/- 21 minutes) (P < .01). The time to thrombus was prolonged by L-arginine (P < .05) and SNP (P < .01). After 3 hours of thrombus formation in coronary arteries, tissue plasminogen activator and heparin were administered intravenously. Thrombi were lysed in 4 (of 8) dogs in the saline group (71 +/- 8 minutes), in 4 (of 8) dogs in the L-NNA group (72 +/- 8 minutes), in 4 (of 6) dogs in the L-arginine group (50 +/- 14 minutes), and in 4 (of 6) dogs in the SNP group (49 +/- 11 minutes) (P > .05). After thrombolysis, coronary artery reocclusion developed in all reperfused dogs in the saline group (30 +/- 8 minutes) and in the L-NNA group (48 +/- 12 minutes), in 3 (of 4) reperfused dogs in the L-arginine group (123 +/- 26 minutes), and in 3 (of 4) reperfused dogs in the SNP group (128 +/- 19 minutes) (P < .01). The ex vivo platelet aggregation induced by collagen was inhibited after in vivo treatment with L-arginine or SNP. CONCLUSIONS: Increasing NO production or giving an NO donor may inhibit platelet aggregation and delay intracoronary thrombus formation and reocclusion after thrombolysis.

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. These are heparin--heparan sulfate, transforming growth factor beta, and nitric oxide. The effect of sympathetic innervation on vascular growth is probably dependent on its interaction with the renin-angiotensin system. In the mesenteric vascular bed, the elevated resistance in the arterial system is present in both the macroarteries and in the more distal microarteries and veins. Changes in resistance arteries include hypertrophy and reduction in outer diameter (remodelling). In the resistance arteries from human essential hypertensives, remodelling is the predominant finding. Long-term treatment with an angiotensin I converting enzyme inhibitor but not with a beta-blocker was effective in reversing this type of vascular change. Studies have suggested that in addition to angiotensin II, endothelin may play a role in vascular remodelling of resistance arteries.

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. Expression of FGFR isoforms was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using primers for the conserved tyrosine kinase (TK) domain followed by Southern blotting with TK insert probes unique to each isoform. The data indicate that FGFR I is the major form of FGF receptor mRNA expressed by proliferating human arterial smooth muscle cells. This strongly suggests that it is this type of FGFR that mediates the signal transduction cascade associated with mitogenesis in proliferating human smooth muscle cells.

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. Lipopolysaccharide-stimulated SHR-derived smooth muscle cells produced less NO. than WKY cells. However the amounts produced in response to interleukin-1 beta were similar for the two cell types. DL-propranolol increased the NO. production in both types of cells exposed to lipopolysaccharide, but had no significant effect on this parameter in WKY-derived cells exposed to interleukin-1 beta. Inclusion of L-arginine during incubations with propranolol had no effect on the levels of NO. produced by either cell type exposed to lipopolysaccharide. The basal production of NO. was enhanced in smooth muscle cells from both normotensive and hypertensive rats when the cells were treated with L-arginine after exposure to interleukin-1 beta. L-Arginine increased the response to DL-propranolol only in the WKY cells. NO. production was depressed by inclusion of NG-nitro-L-arginine during incubations in both cell types regardless of the treatment regime used to induce NO. synthase activity. The results suggest that DL-propranolol may induce the production of NO. by cultured smooth muscle cells exposed to cytokines.

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. GTP:cyclohydrolase is the regulatory enzyme for de novo tetrahydrobiopterin synthesis, and the latter is a required cofactor for NO synthase activity. Treatment of smooth muscle cells with forskolin induced GTP:cyclohydrolase mRNA expression, and simultaneous treatment of cells with forskolin and phorbol esters elicited NO production. Angiotensin II and arginine-vasopressin, acknowledged agonists for protein kinase C, elicited production of NO by forskolin-treated smooth muscle cells. These observations confirm the importance of GTP:cyclohydrolase activity for NO production by cultured smooth muscle cells and implicate both adenylyl cyclase and protein kinase C in this process.

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. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.

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. In addition, endothelial cells release endothelium-derived hyperpolarizing factor, which regulates potassium-channel opening in vascular smooth muscle. The chemical nature of this molecule remains to be elucidated. Many of the physiologic stimulants for endothelium-derived relaxing factor production are released by aggregating platelets, and the significance of the endothelium's vasoprotective role becomes apparent when the mechanisms and consequences of platelet agglutination are studied. Damage to the endothelium, however minor, results in the loss of this protective function and is associated with an impaired response to serotonin of G-protein coupled receptors. In the presence of risk factors such as elevated serum cholesterol, the consequences of an impaired endothelial function are greatly enhanced. Age-related changes in endothelial responsiveness may account for the prevalence of cardiovascular disease in human beings over the age of 30 years.

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 inhibitors appear to comprise at least 3 distinct types of molecules: heparin/heparan sulfate; transforming growth factor beta; and nitric oxide. Each molecule inhibits growth of cultured smooth muscle cells by mechanisms that remain to be elucidated and are discussed in this communication. Heparin/heparan sulfate is the most thoroughly characterized of the 3, and has been used for clinical intervention to prevent restenosis. Transforming growth factor beta exhibits bimodal activity on growth, acting as a stimulant at low levels and as an inhibitor at elevated concentrations. Nitric oxide mediated vasorelaxation is dependent upon activation of soluble guanylate cyclase. Because elevation of cyclic guanosine monophosphate in smooth muscle cells depresses their proliferation, nitric oxide would appear to possess the properties necessary to inhibit vascular smooth muscle cell proliferation.

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. Peptide growth factors such as fibroblast growth factor, platelet-derived growth factor and transforming growth factor beta 1 and the protease thrombin have been shown to modulate the production NO by cytokine-treated smooth muscle cells. These peptide agonists also regulated the induction of NO synthase and GTP: cyclohydrolase mRNA expression.

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. Contractions to 5-hydroxytryptamine were larger in preparations from hypertensive than normotensive animals. Treatment with interleukin-1 beta for 6 h reduced the responsiveness to both vasoconstrictors in a concentration-dependent manner. The depression was more pronounced in rings from spontaneously hypertensive rats: the threshold concentration of the cytokine was lower, and its maximal effect greater. Nitro-L-arginine prevented the inhibitory effect of interleukin-1 beta. The cytokine evoked a time-dependent loss of tone in phenylephrine-contracted rings with the same time of onset in both strains. However, the decay of tension was more pronounced in aortae from hypertensive than normotensive rats. In aortae from both strains, the decay was potentiated by L-arginine, but not D-arginine. Interleukin-1 beta elicited greater concentration-dependent productions of cyclic GMP and nitrite in rings from spontaneously hypertensive than from Wistar-Kyoto rats, and these were inhibited by methylene blue and nitro-L-arginine, respectively. The concentration-relaxation curves to 3-morpholino-sydnonimine were moderately, but significantly, shifted to the left in aortae from spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)