Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells

The effect of glucocorticoids on collagen synthesis was examined in cultured bovine aortic smooth muscle (BASM) cells. BASM cells treated with 0.1 microM dexamethasone during their proliferative phase (11 d) were labeled with [3H]proline for 24 h, and the acid-precipitable material was incubated with bacterial collagenase. Dexamethasone produced an approximate twofold increase in the incorporation of proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) in the cell layer and medium. The stimulation was present in both primary mass cultures and cloned BASM. An increase in CDP and NCP was detected at 0.1 nM, while maximal stimulation occurred at 0.1 microM. Only cells exposed to dexamethasone during their log phase of growth (1-6 d after plating) showed the increase in CDP and NCP when labeled 11 d after plating. The stimulatory effect was observed in BASM cells treated with the natural bovine glucocorticoid, cortisol, dexamethasone, and testosterone, but was absent in cells treated with aldosterone, corticosterone, cholesterol, 17 beta-estradiol, and progesterone. The increase in CDP and NCP was absent in cells treated with the inactive glucocorticoid, epicortisol, and totally abolished by the antagonist, 17 alpha-hydroxyprogesterone, suggesting that the response was mediated by specific cytoplasmic glucocorticoid receptors. Dexamethasone-treated BASM cells showed a 4.5-fold increase in the specific activity of intracellular proline, which was the result of a twofold increase in the uptake of proline and depletion of the total proline pool. After normalizing for specific activity, dexamethasone produced a 2.4- and 2.8-fold increase in the rate of collagen and NCP synthesis, respectively. Cells treated with dexamethasone secreted 1.7- fold more collagen protein in 24 h compared to control cultures. The BASM cells secreted 70% Type I and 30% Type III collagen into the media as assessed by two-dimensional gel electrophoresis. The ratio of these two types was not altered by dexamethasone. The results of the present study demonstrate that glucocorticoids can act directly on vascular smooth muscle cells to increase the synthesis and secretion of collagen and NCP.     

Modulation of collagen synthesis by tumor necrosis factor alpha in cultured vascular smooth muscle cells

Collagen synthesis in vascular smooth muscle cells (SMCs) after exposure to tumor necrosis factor alpha (TNF-alpha) was investigated using a culture system. The synthesis of collagenase-digestible proteins (CDP) and noncollagenous proteins (NCP) was evaluated by the [3H]proline incorporation. It was shown that TNF-alpha markedly suppresses the incorporation of [3H]proline into both CDP and NCP in confluent cultures of SMCs but not in sparse cultures of the cells. Such a marked suppression by TNF-alpha was not observed in confluent bovine aortic endothelial cells and human fibroblastic IMR-90 cells. In confluent SMCs, the synthesis of CDP was more strongly inhibited by TNF-alpha than that of NCP. When the CDP synthesis was stimulated by transforming growth factor beta, TNF-alpha suppressed the stimulation in both confluent and sparse SMCs. Human SMCs synthesized types I, III, IV and V collagens; TNF-alpha markedly decreased the relative proportion of types IV and V. It was therefore suggested that TNF-alpha modulates the collagen synthesis by SMCs depending on their cell density and modifies the formation of atherosclerotic lesions.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca²⁺ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca²⁺ as well as PG synthesis. The increase of cytosolic Ca²⁺ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca²⁺ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGIA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca²⁺, whereas TMB-8 (10 μM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca²⁺, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca²⁺.These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca²⁺ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca²⁺ and on the other hand, PG synthesis.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.     

Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time- and dose-dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5-hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C-activating phorbol ester, phorbol 12 myristate 13-acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.     

Intercellular communication in cultured human vascular smooth muscle cells

Intercellular communication through gap junction channelsplays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,capacitance vessel). Northern and Western blots documented the presenceof connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA incultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority ofrecords, in both vessels, single-channel activity was dominated by amain-state conductance of 120 pS, with subconducting events comprisingless than 10% of the amplitude histograms. However, some recordsshowed "atypical" unitary events that had a conductance similar toCx40 (~140-160 pS), but gating behavior like that of Cx43. Assuch, it is conceivable that the presence and coexpression of Cx40 andCx43 in IMA and SV myocytes may result in heteromeric channelformation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

     

Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells

We explored the hypothesis that discrepancies in the literature concerning the nature of myosin expression in cultured smooth muscle cells are due to the appearance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two MHCs in intact smooth muscles (SM1 and SM2) which differ by less than 2% in molecular weight (Rovner, A. S., Thompson, M. M., and Murphy, R. A. (1986) Am. J. Physiol. 250, C861-C870). Myosin-containing homogenates of rat aorta cells in primary culture were electrophoresed on this gel system, and Western blots were performed using smooth muscle-specific and nonmuscle-specific myosin antibodies. Subconfluent, rapidly proliferating cultures contained a form of heavy chain not found in rat aorta cells in vivo (NM) with electrophoretic mobility and antigenicity identical to the single unique heavy chain seen in nonmuscle cells. Moreover, these cultures expressed almost none of the smooth muscle heavy chains. In contrast, postconfluent growth-arrested cultures expressed increased levels of the two smooth muscle heavy chains, along with large amounts of NM. Analysis of cultures pulsed with [35S] methionine indicated that subconfluent cells were synthesizing almost exclusively NM, whereas postconfluent cells synthesized SM1 and SM2 as well as larger amounts of NM. Similar patterns of MHC content and synthesis were found in subconfluent and postconfluent passaged cells. These results show that cultured vascular smooth muscle cells undergo differential expression of smooth muscle- and nonmuscle-specific MHC forms with changes in their growth state, which appear to parallel changes in expression of the smooth muscle and nonmuscle forms of actin (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352). The reappearance of the smooth muscle MHCs in postconfluent cells suggests that density-related growth arrest promotes cytodifferentiation, but the continued expression of the nonmuscle MHC form in these smooth muscle cells indicates that other factors are required to induce the fully differentiated state while in culture.     

Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation

The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.     

Growth and differentiation of the vascular smooth muscle and endothelial cells cultured on fluorine ion-implanted polystyrene

The rat vascular (SMCs) and bovine endothelial cells (BECs) were cultured on conventional or fluorine ion-implanted polystyrene (5 x 10(12) and 5 x 10(14) fluorine ions/cm2). The cells grown on the implanted growth supports showed better adherence, higher volume and higher total protein content. The immunocytochemical analysis revealed that SMCs contained more of the cytoskeletal vimentin and the vascular SMC-specific alpha-actin as well as several cell adhesion-mediating molecules (vinculin, talin, alpha(v)-integrin and ICAM-1). In BECs, only the content of vimentin and talin increased, while expression of ICAM-1 was unchanged. The data suggest that cells on the ion implanted polymers could be more viable and that increased expression of some adhesion molecules mediating interactions with the host immune system is cell type-dependent.     

Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells

Angiotensin II stimulates sequential phospholipase C-mediated hydrolysis of initially the polyphosphoinositides and subsequently phosphatidylinositol (PI) in cultured rat aortic smooth muscle cells resulting in biphasic, sustained formation of diacylglycerol (DG). The mechanisms underlying this delayed induction of sustained DG accumulation are unknown but may be related to cellular events including processing of the angiotensin II receptor-ligand complex. In the present study, we characterized the kinetics of angiotensin II receptor sequestration and studied the effects of interventions which interfere with receptor processing on the pattern of angiotensin II-induced DG formation and phosphoinositide hydrolysis. Conversion of the angiotensin II receptor to an acid-resistant form was temperature-dependent, with half-times of 1.5 min at 37 degrees C and 7 min at 19 degrees C. Reducing the temperature to 25 or 19 degrees C caused a marked temporal separation between the two phases of DG accumulation. There was a close temporal correlation between the effect of temperature on receptor sequestration and on sustained DG accumulation. Furthermore, phenylarsine oxide (5 min, 10 microM), which inhibited angiotensin II receptor internalization, also selectively inhibited the sustained phase of DG accumulation (81 +/- 6% inhibition). Monensin and chloroquine, which interfere with receptor processing through the lysosomal-degradative pathway, had no effect on angiotensin II-induced DG formation in these cells, suggesting that the processing event important to hormonally induced sustained DG accumulation occurs early in the internalization pathway, probably at the level of the plasma membrane. Moreover, the acid-resistant state of the angiotensin II receptor-ligand complex retained its ability to signal, since removal of the surface signal by competitive antagonism with Sar1-Ile8-angiotensin II or acid-wash only slowly reversed accumulation of DG and depression of total cell calcium. These experiments support our previous observation that the initial and sustained phases of angiotensin II-induced diacylglycerol formation in vascular smooth muscle are differentially controlled and suggest that an early event in the cellular processing of the angiotensin II-receptor complex is essential to maintenance of DG accumulation.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells

Cultured vascular smooth muscle cells from bovine and rat thoracic aortae and from human omental vessels have been examined for cellular responses to endothelin. In myo-[3H]-inositol-prelabelled cells endothelin induced a rapid (within 30 sec) and protracted increase of [3H]-inositol content in inositol bis- and tris-phosphates. Concomitantly, significant polyphosphoinositide hydrolysis occurred within 30 sec. Accumulation of [3H]-inositol monophosphate and hydrolysis of phosphatidylinositol were delayed. In cells prelabelled with [3H]-arachidonic acid endothelin promoted rapid production of [3H]-diacylglycerol which decayed slowly toward control values after reaching maximum levels (1-2 min). Half-maximally effective concentrations of endothelin for all these cellular responses were comparable (approximately 3-7 nM) and not significantly different between the vascular cell isolates. The involvement of the phospholipase C-signal transduction pathway in mediating endothelin-induced vasoconstriction is invoked.     

Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells

The migration of vascular smooth muscle cells (VSMCs) is a principal factor for the development and progression of vascular diseases. In addition, phenotypic alteration from the contractile (differentiated) to the synthetic (dedifferentiated) state and a proteolytic process in the form of extra cellular matrix degradation are necessary for SMC invasion. The actual mechanism leading to the focal degradation of basement membrane matrix components and, hence, SMC migration within the tissue itself is, however, unclear. In response to phorbol ester [phorbol-12,13-dibutyrate (PDBu)], VSMCs in culture form podosomes, dynamic organelles critical for cell adhesion and substrate degradation that are typically found in invasive cells and cells that cross tissue boundaries. Here, we show that PDBu-stimulated VSMCs resorb the extracellular matrix at the sites of podosomes. Podosome formation correlates with an increased polarization of VSMCs on fibronectin- or collagen-coated flexible substrates in addition to a concomitant induction of cell motility. VSMCs embedded in reconstituted basement membrane support adopt the typical spindle-shaped morphology of differentiated SMCs in vivo and, after PDBu treatment, form peripheral lamellipodia and podosomes around their matrix-contacting surface. Our findings demonstrate that podosome formation is the potential mechanism underlying the ability of VSMCs to traverse the surrounding basement membrane and escape the barrier of the tunica media in vascular diseases.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells

Adipose tissue-derived stromal cells (ADSC) have previously been shown to possess stem cell properties such as transdifferentiation and self-renewal. Because future clinical applications are likely to use these adult stem cells in an autologous fashion, we wished to establish and characterize rat ADSC for pre-clinical tests. In the present study, we showed that rat ADSC expressed stem cell markers CD34 and STRO-1 at passage 1 but only STRO-1 at passage 3. These cells could also be induced to differentiate into adipocytes, smooth muscle cells, and neuron-like cells, the latter of which expressed neuronal markers S100, nestin, and NF70. Isobutylmethylxanthine (IBMX), indomethacin (INDO), and insulin were the active ingredients in a previously established neural induction medium (NIM); however, here we showed that IBMX alone was as effective as NIM in the induction of morphological changes as well as neuronal marker expression. Finally, we showed that vascular smooth muscle cells could also be induced by either NIM or IBMX to differentiate into neuron-like cells that expressed NF70.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of TNF to the culture medium for 24-48 h resulted in an increase of [3H]oleic acid uptake and esterification into lipids. The effect could be seen already with 0.3 ng/ml and was maximal with 30 ng/ml. The effect of TNF was mainly on the incorporation of [3H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3H]cholesteryl ester. In the human SMC there was a 40% increase in [3H]oleic acid into total lipids, while the rise in [3H]triacylglycerol ranged between 60-90%. TNF did not modulate cellular triacyglycerol synthesis in cultured mouse peritoneal macrophages. Since TNF was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triacylglycerol accumulation.     

Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.