Increased sulphation level and altered composition of glycosaminoglycans synthesized by cultured smooth muscle cells in the presence of beta-D-xylosides.

Cultured rat and bovine smooth muscle cells incorporated more 35SO4 into macromolecular glycosaminoglycans in the presence of beta-D-xylosides than in their absence. More than 90% of the xyloside-initiated glycosaminoglycans were secreted rapidly into the culture medium and were more highly sulphated than glycosaminoglycans polymerized on core protein. The increased extents of sulphation were associated with increased synthesis of dermatan sulphate and a decrease in that of nitrous acid-sensitive glycosaminoglycans.

Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats.

Aortic smooth muscle cells from spontaneously hypertensive rats (SHR) exhibit inappropriate proliferation characteristics in culture that suggest a modified response to serum mitogens or growth factors. The present study compares vascular smooth muscle cells from SHR and normotensive Wistar-Kyoto (WKY) rats with respect to their proliferative and functional response to growth factors. Specific attention was focused on the interaction of these vascular smooth muscle cells with epidermal growth factor. An increased growth rate of vascular smooth muscle cells from SHR (vs. WKY rats) was observed when cells were cultured in the presence of serum (10% and 0.5%), but not under serum-free conditions. The additional presence of low serum concentrations (0.5%) was required for epidermal growth factor to elicit a proliferative response, whereupon smooth muscle cells from SHR displayed an increased (vs. WKY rats) growth rate. Saturation binding of [125I]epidermal growth factor to intact smooth muscle cells indicated a twofold increase in receptor density in SHR-derived cells (p less than 0.001 vs. WKY rats) without an alteration in affinity for the growth factor. Cells derived from SHR also exhibited greater functional responsiveness to epidermal growth factor when compared with smooth muscle cells from WKY rats as evidenced by amplifications of both S6 kinase activation, phosphoinositide catabolism, elevation of intracellular pH, and DNA synthesis (nuclear labeling). We conclude that increased responsiveness of SHR-derived smooth muscle cells to epidermal growth factor could contribute to alterations in vascular smooth muscle growth and tone that may be fundamental to the pathogenesis of hypertension and atherosclerosis.

Enhanced responsiveness to angiotensin II in vascular smooth muscle cells from spontaneously hypertensive rats is not associated with alterations in protein kinase C.

This study compares vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats with respect to protein kinase C and intracellular responses to angiotensin II (Ang II). Ang II-induced degradation of polyphosphoinositides and accumulation of inositol di- and tris-phosphates was enhanced (approximately twofold) in hypertensive-derived cells, without a change (vs. normotensive-derived cells) in half-maximally effective concentrations of Ang II. Intracellular pH (approximately 6.6) was comparable between both cell isolates at quiescence, but alkalinization induced by Ang II, serum, or phorbol ester was greater (delta 0.1-0.2 pH units) for hypertensive-derived cells. For both cell types, the intracellular pH response to these agonists was prevented in the presence of Na+-H+ exchange inhibitors. S6 kinase activation induced by Ang II was enhanced (approximately twofold) in hypertensive-derived cells, whereas activation in response to serum or 12-O-tetradecanoylphorbol 13-acetate did not differ significantly between the two cell types. Quantitation of protein kinase C by immunoblotting and [3H]phorbol dibutyrate binding procedures revealed no differences between the two smooth muscle cell isolates (at quiescence or in the presence of serum) with respect to either total amounts or subcellular distribution. Sensitivity of protein kinase C to phorbol ester was apparently also not different between the two cell types, as assessed from dose-dependent (phorbol ester) S6 kinase activation profiles. Phorbol ester caused a similar subcellular redistribution of [3H]phorbol dibutyrate binding in the two cell isolates, but for both, minimal (10%) translocation occurred in response to Ang II. The data suggest that enhanced agonist responsiveness in vascular smooth muscle cells is unlikely to involve alterations in protein kinase C.

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. Results obtained from pharmaceutical intervention in humans and animals of these systems strongly support this hypothesis. In addition to their vasoconstrictor properties, angiotensin II and endothelin-1 act as potent biologic effectors. In vitro, both vasoconstrictor peptides appear to modulate the activity of autocrine feedback loops in vascular smooth muscle cells. The activity of these feedback loops in vivo may represent a central mechanism for regulation and phenotypic differentiation of this cell type. The most well-established autocrine feedback loops of vascular smooth muscle cells are constituted by platelet-derived growth factor and transforming growth factor-beta, both of which are influenced by the action of angiotensin II and endothelin-1. The effects of the peptide vasoconstrictors on the (auto-) regulated feedback loops are of long-term structural importance, since both vasoconstrictors (via autocrine growth modulators) may influence the composition of the extracellular matrix of vascular smooth muscle cells. This includes effects on the synthesis and secretion of thrombospondin, fibronectin, tenascin, etc. The secretion of extracellular matrix glycoproteins themselves and incorporation into extracellular matrix in vitro appear to be linked to the activity of the autocrine feedback loops: e.g., stimulation of thrombospondin mRNA results in secretion of the glycoprotein only in the concomitant presence of exogenous platelet-derived growth factor, whereas the expression of fibronectin and tenascin may be directed by transforming growth factor-beta. The influence of angiotensin II and endothelin-1 on vascular smooth muscle cell surface receptor expression may represent a secondary mode of action of these vasoconstrictor peptides. Endothelin-1, for instance, can rapidly down-regulate platelet-derived growth factor-alpha receptor mRNA and both angiotensin II and endothelin-1, via induction of transforming growth factor-beta, may interrupt the platelet-derived growth factor based autocrine feedback loop. In vivo, the highly complex interactions between local and systemic vasoconstrictor production, autoregulated feedback loops, and extracellular matrix (which also serves as a reservoir for growth and differentiation modulators) are central to vessel homeostasis.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Atrial natriuretic peptide: binding and cyclic GMP response in cultured vascular smooth muscle cells from spontaneously hypertensive rats.

Atrial natriuretic peptide (ANP) is vasodilatory and natriuretic, but whereas increased plasma ANP levels occur in spontaneously hypertensive rats, their elevated vascular resistance suggests inappropriate target tissue responsiveness to ANP. This study examines ANP-receptor binding properties (at 25 degrees C and 4 degrees C) in cultured vascular aortic smooth muscle cells from spontaneously hypertensive (SHR) and control Wistar-Kyoto (WKY) rats. [I125]-human ANP saturation (0.0625-12.0 nmol) profiles were analyzed using nonlinear regression (LIGAND). Vascular smooth muscle cells from WKY possessed both high affinity (KD1 0.3 nmol; R1 33 fmol/10(5) cells) and low affinity (KD2 15 nmol; R2 400 fmol/10(5) cells) binding sites for ANP. In contrast, for smooth muscle cells from SHR, two receptor forms could not be resolved using identical analytical protocols. Parameter estimates at 25 degrees C and 4 degrees C were not different for either SHR or WKY. The number of receptors for SHR (Bmax approximately 100 fmol/10(5) cells) was lower than the total number of receptors for WKY (high plus low affinity approximately 430 fmol/10(5) cells). The intermediary KD value (approximately 1.0 nmol) for ANP binding in SHR suggests an ANP-receptor interconversion from high affinity to low affinity in smooth muscle cells from SHR. Competition-binding experiments also revealed a decreased affinity for ANP in SHR-derived smooth muscle cells. The cyclic GMP response (intracellular accumulation and extracellular levels) was decreased in SHR smooth muscle cells compared to WKY, although this difference was evident only after prolonged (one hour) stimulation with ANP. Our data indicate a reduced sustained vascular responsiveness to ANP in hypertension.

Angiotensin-induced growth related metabolism is activated in cultured smooth muscle cells from spontaneously hypertensive rats and Wistar-Kyoto rats.

Smooth muscle cells from spontaneously hypertensive rats (SHR) proliferate in culture faster than those isolated from sex- and age-matched Wistar-Kyoto (WKY) animals. There was no difference in the kinetics of S6 kinase activation in the two cultures, but later metabolic events associated with proliferation were stimulated earlier in SHR cells than in WKY, eg, activation of ornithine decarboxylase. Both cell types elaborated an extensive extra-cellular matrix in culture composed of a different blend of connective tissue macromolecules. Matrix material from SHR cells was more stimulatory to growth of WKY cultures than their own matrices. Angiotensin stimulated the growth and synthesis of extra-cellular matrix material in SHR more than in WKY derived vascular smooth muscle cell cultures.

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)

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.

The induction of nitric oxide synthase activity is inhibited by TGF-beta 1, PDGFAB and PDGFBB in vascular smooth muscle cells.

The effect of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor (PDGF) was investigated on the induction of nitric oxide synthase activity caused by interleukin-1 beta in cultured smooth muscle cells from rat aorta. TGF-beta 1, PDGFAB and PDGFBB but not PDGFAA inhibited in a concentration-dependent manner the production of nitrite, an oxidation product of nitric oxide, evoked by interleukin-1 beta. The growth factors alone did not stimulate the release of nitrite. The addition of interleukin-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated human washed platelets inhibited the aggregation evoked by thrombin whereas no effect was observed with untreated cells. Platelet aggregation was not inhibited by smooth muscle cells that had been pretreated with interleukin-1 beta in combination with either TGF-beta 1, PDGFAB or PDGFBB but not with PDGFAA. These observations demonstrate that platelet-derived products such as TGF-beta and PDGFs inhibit the induction of nitric oxide synthase activity in vascular smooth muscle cells.

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.

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.

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.

Increased proliferation fate and phosphoinositide turnover in cultured smooth muscle cells from spontaneously hypertensive rats.

We cultured smooth muscle cells from matched sections of thoracic aortas of 20-week-old, male, spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats and compared their rates of proliferation and phosphoinositide metabolism. After seeding at initially identical densities, smooth muscle cells from SHR grow at significantly faster (P less than 0.001) rates than those from WKY rats, so that after 10 days in culture, the cell density of SHR-derived cells (3.10 +/- 0.32 x 10(5) cells/cm2) was double that of WKY-derived cells (1.59 +/- 0.28 x 10(5) cells/cm2). Following prolonged steady-state labelling in culture, incorporation of [3H]-myo-inositol into phosphatidylinositolbiphosphate (PIP2) was greater (P less than 0.01) in SHR smooth muscle cells (10.3 +/- 1.4%) than in those from WKY (6.9 +/- 1.4%). Incorporation of [3H]-myo-inositol into inositol triphosphate was also increased (P less than 0.01) in SHR (20.2 +/- 7.3%) relative to WKY cells (11.5 +/- 5.2%), while that into glycerophosphoinositol was lower (P less than 0.01) in SHR (46.5 +/- 8.0%) than in WKY (51.3 +/- 7.8%). Enhanced smooth muscle cell growth and altered phosphoinositide metabolism may be causally or consequentially involved in enhanced vasoconstriction and vascular hypertrophy in SHR.