The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.     

Effect of serum on vascular smooth muscle function

We investigated the in vitro and in vivo effects of KT6352, a derivative of indolocarbazole compound, on murine megakaryocytopoiesis. When serum-free megakaryocyte (Meg) colony assay was performed with 100 U/mL of recombinant mouse interleukin-3 (rmIL-3), the addition of 1x10(-11)M to 1x10(-9)M of KT6352 increased the number of Meg colonies. An additional increase of Meg colonies by KT6352 was observed in the serum-free culture containing rmIL-3 plus recombinant mouse interleukin-6 or rmIL-3 plus recombinant mouse stem cell factor. KT6352 did not stimulate Meg colony formation without rmIL-3. When KT6352 was administered intraperitoneally to normal BALB/c male mice at a dose of 10 mg/kg daily for 5 consecutive days, a 2.1-fold increase in the platelet count was observed on day 14, and the prolonged thrombocytopoiesis was detectable from 9 to 27 days after KT6352 administration. A marked increase in the white blood cell count was also observed from 5 to 14 days after KT6352 treatment. Before the gradual increase of platelet counts, 8 days after KT6352 administration, a marked increase in the number of colony-forming units of megakaryocytes (CFU-Megs) in bone marrow and spleen was observed, and a substantial increase in the number of splenic CFU-Megs was observed 14 and 23 days after KT6352 administration. Bone marrow Meg ploidy analysis by two-color flow cytometry showed a shift in the modal ploidy class from 16 to 32 and an increase in the frequency of 64 cells in KT6352-treated mice. These results suggest a possible therapeutic benefit of KT6352 in the management of thrombocytopenia.     

Identification of distinct molecular phenotypes in cultured gastrointestinal smooth muscle cells

BACKGROUND & AIMS: Cultured gastrointestinal smooth muscle cells have been shown to dedifferentiate and reinitiate their myogenic program in vitro. The aim of this study was to determine whether the cellular phenotypes observed in vitro were similar to those previously characterized in vivo. METHODS: Differential isoactin expression was examined in primary cultures of intestinal smooth muscle cells (ISMCs) by Northern blot and immunohistochemical analysis. Cellular phenotype was determined for cultured ISMCs grown at high density, at low density, in the presence and absence of serum supplementation, and on several distinct substrates including collagen type IV, laminin, fibronectin, and plastic. RESULTS: The unique patterns of isoactin protein and gene expression observed in cultured ISMCs indicate that distinct cellular phenotypes were present in vitro. The production and maintenance of these distinct smooth muscle cell phenotypes was dependent on cell density, serum supplementation, and substrate used. CONCLUSIONS: Cultured ISMCs appear to recapitulate a portion of their in vivo myogenic program in vitro, providing a unique opportunity for the molecular mechanisms controlling gastrointestinal smooth muscle myogenesis and pathogenesis to begin to be identified.     

Progesterone inhibits arterial smooth muscle cell proliferation

Mortality from atherosclerotic cardiovascular disease is lower in premenopausal women than in age-matched men. It is also lower in postmenopausal women who take estrogens and progestins together rather than estrogens alone. Progesterone receptors were detected in human and rat aortic smooth muscle cells in vivo and in vitro (in subculture). We examined the effect of progesterone on proliferation of smooth muscle cells, important constituents of atherosclerotic plaques. Progesterone at physiologic levels inhibited DNA synthesis and proliferation in these cells in a dose-dependent manner, and pretreatment with the progesterone receptor antagonist RU486 blocked inhibition. Cyclin A and E messenger RNA levels decreased after progesterone treatment but those of cyclin B and D1 did not change. This cell cycle-dependent inhibition of arterial smooth muscle cell proliferation by progesterone may represent a mechanism for the hormone's protective effect against atherosclerosis.     

Molecular control of vascular smooth muscle cell differentiation

Changes in the differentiated state of the vascular smooth muscle cell (SMC) including enhanced growth responsiveness, altered lipid metabolism, and increased matrix production are known to play a key role in development of atherosclerotic disease. As such, there has been extensive interest in understanding the molecular mechanisms and factors that regulate differentiation of vascular SMC, and how this regulation might be disrupted in vascular disease. Key questions include determination of mechanisms that control the coordinate expression of genes required for the differentiated function of the smooth muscle cell, and determination as to how these regulatory processes are influenced by local environmental cues known to be important to control of smooth muscle differentiation. Of particular interest, a number of common cis regulatory elements including highly conserved CArG [CC(A/T)6GG] motifs or CArG-like motifs and a TGF beta control element have been identified in the promoters of virtually all smooth muscle differentiation marker genes characterized to date including smooth muscle alpha-actin, smooth muscle myosin heavy chain, telokin, and SM22 alpha and shown to be required for expression of these genes both in vivo and in vitro. In addition, studies have identified a number of trans factors that interact with these cis elements, and shown how the expression or activity of these factors is modified by local environmental cues such as contractile agonists that are known to influence differentiation of smooth muscle.     

The cytoskeleton of the vertebrate smooth muscle cell

Four experiments were conducted to determine the effects of dopamine (DA) antagonists and DA depletions on progressive-ratio responding for food reinforcement. On this schedule, ratio requirement increased by one response after each reinforcer was obtained, and rats were tested in 30-min sessions. Response rates and highest ratio completed were reduced in a dose-related manner by systemic injections of the D1 antagonist SCH 23390, and also by the D2 antagonists haloperidol and raclopride. Drug-treated rats also showed reductions in time to complete the last ratio, demonstrating that they had stopped responding before the end of the session. DA depletions produced by injections of 6-OHDA directly into the nucleus accumbens substantially decreased both the number of responses and the highest ratio completed. The deficits in response number and highest ratio completed induced by DA depletions persisted through the first 3 weeks of postsurgical testing, with some recovery by the fourth week. However, the deficits resulting from dopamine depletions were largely a manifestation of a decrease in response rate; although time to complete the last ratio was significantly reduced by dopamine depletions in the first few days of testing, rats recovered on this measure by the fifth day after surgery. Although previous work has shown that performance on several schedules (e.g., continuous, low value ratios, variable interval) is relatively unaffected by accumbens DA depletions, the present data demonstrate that such depletions do produce a substantial and persistent impairment of progressive ratio response output. Rats with accumbens DA depletions appear to have deficits in maintaining the high work output necessary for responding at large ratio values. The relative sparing of responding on some simple schedules, together with the present progressive ratio results, suggest that rats with accumbens DA depletions remain directed toward the acquisition and consumption of food, but they show deficits in work output for food.     

Nitrergic innervation and relaxant response of rectal circular smooth muscle

We report a case of an anomalous right coronary artery arising from the morphological left sinus of Valsalva in a patient with Kartagener's syndrome. Literature review has revealed only a small number of cases of anomalous coronary arteries in patients with dextrocardia and none previously reported in Kartagener's syndrome.     

Control of vascular smooth muscle cell growth in fowl

In adult domestic fowl, angiotensin (ANG) receptors are present in the vascular smooth muscles (VSM) and in the endothelium, mediating vasorelaxation via endothelium-derived relaxing factor/cGMP. ANG II-induced relaxation is minor in chicks and becomes more marked as they mature but diminishes in adult birds, whereas ANG II neither relaxes nor contracts endothelium-denuded aortae from mature chickens. The present study examines in cultured fowl aortic SM cells whether (1) ANG II stimulates or inhibits VSM cell growth and, if so, whether this growth-stimulatory or -inhibitory effect changes with maturation/aging, and (2) S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, and cGMP attenuate the basal or stimulated VSM cell growth. [Asp1, Val5]ANG II (native fowl ANG II, 10(-6) M) markedly increased (increase from vehicle control, 226.5%; P < 0.01) [3H]thymidine (Thd) incorporation into DNA of quiescent VSM cells (first subculture) from 6-week-old chicks. This growth-stimulating effect was reduced with age (41.4, 29.6, and 3.2% at 9, 19, and 43 weeks of age, respectively). In contrast, platelet-derived growth factor (PDGF, 20 ng/ml) increased [3H]Thd incorporation similarly in chicks, pullets, and hens. Furthermore, ANG II significantly (45.9%, P < 0.01) attenuated the growth-promoting effect of fetal calf serum in cultured VSM cells from 6-week-old chicks. This inhibitory effect also decreased in older birds. ANG II showed neither a growth-stimulatory nor -inhibitory effect in cultured neointimal cells. SNAP attenuated dose dependently (20-60 microM) the basal and PDGF-induced VSM cell growth, whereas cGMP inhibited basal growth only at a high dose (100 microM). These results indicate that in fowl VSM cells, ANG II is mitogenic and antimitogenic in chicks but not in mature birds, suggesting that phenotypic modulation occurs in the ANG receptors/signaling mechanism with maturation/age or in neointimal cells, whereas the mitogenic mechanism via PDGF remains in both young and mature birds.     

Monocyte-vascular smooth muscle cell interaction enhances nitric oxide production

OBJECTIVE: The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis. We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression. METHODS: NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting. RESULTS: Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha. The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody. CONCLUSIONS: The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.     

Heparin inhibits human coronary artery smooth muscle cell migration

Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis following vascular injury in experimental studies, but the clinical trials of heparin in coronary balloon angioplasty have been negative. The current study, therefore, examined the effect of heparin on basal or stimulated migration by serum and platelet-derived growth factor (PDGF)-BB in cultured human coronary artery smooth muscle cells (SMCs) by Boyden's chamber method. In addition, the reversibility of the heparin effect on human coronary artery SMC migration was examined. Fetal calf serum (FCS) and PDGF-BB stimulated SMC migration in a concentration-dependent manner. Heparin in moderate to high concentration (10 to 100 U/mL) exhibited concentration-related inhibition of FCS- and PDGF-BB-stimulated SMC migration; however, a low concentration (1 U/mL) of heparin had no inhibitory effects. Heparin also had weak inhibitory effects on nonstimulated SMC migration. The SMCs that were exposed to a high concentration (100 U/mL) of heparin for 6 hours were capable of migrating after a short lag period of removal of heparin from the culture medium. These SMCs also showed recovery of responses to FCS and PDGF-BB by migrating significantly greater than the nonstimulated level. Furthermore, heparin-containing medium did not contain detached cells. These results indicate that heparin inhibits human coronary artery SMC migration, especially when stimulated by FCS or PDGF-BB, and that this inhibitory effect of heparin is reversible and not simply a function of killing cells.     

Effect of docosahexaenoic acid on smooth muscle cell functions

There is increasing evidence that fish oil-enriched diets attenuate the progression of several types of human and experimental renal, intestinal and cardiovascular disorders, including hypertension. Docosahexaenoic acid (DHA), may be one of the active biological component. We previously reported that dietary DHA suppressed the progression of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). The purpose of this study is to clarify the in vitro effect of DHA on cultured smooth muscle cell functions such as cell growth, hypertrophy, NO release, and intracellular Ca2+ metabolism, which are involved in the regulatory mechanisms of vascular tone. Addition of DHA to the culture medium of aortic smooth muscle cells isolated from SHRSP and normotensive Wistar Kyoto rats (WKY) had no significant effects on cell growth or on cell hypertrophy induced by angiotensin II as measured by flow cytometry. DHA had no stimulatory effect on interleukin-1beta (10 ng/ml)-induced nitric oxide release from smooth muscle cells of SHRSP, but rather slightly inhibited it. However, the treatment of smooth muscle cells with DHA (30 microM) for 2 days significantly suppressed the increase in intracellular Ca2+ concentration induced by angiotensin II, but not by thapsigargin. This was due to the suppression of Ca2+ influx, as determined by Mn2+ influx experiment. These results indicate that DHA specifically suppresses Ca2+ mobilization into smooth muscle cells. This may be one of the mechanisms by which dietary DHA prevents the development of hypertension in SHRSP.     

Control of smooth muscle cell proliferation by psoralen photochemotherapy

PURPOSE: Restenosis after balloon angioplasty or the intimal hyperplasia occurring at distal anastomoses of bypass grafts severely limits the long-term therapy for peripheral vascular disease. The aim of this study was to evaluate the application of psoralen photochemotherapy with ultraviolet A (UVA)-activated 8-methoxypsoralen (8-MOP) to suppress smooth muscle cell (SMC) proliferation in vitro by the formation of 8-MOP-DNA monoadducts and interstrand cross-links to inhibit DNA synthesis. METHODS: Bovine aorta SMC (2 x 10(4)/cm2) were treated with 8-MOP (0 to 1000 ng/ml) for 30 minutes, followed by UVA (2 joule/cm2) to determine the dose of 8-MOP and UVA that inhibits SMC proliferation. RESULTS: The results show that 8-MOP in the range 30 to 1000 ng/ml in combination with 2 joule/cm2 UVA inhibited SMC proliferation by 40% to 60% 3 days after treatment. In time course studies the growth of SMC treated with 100 ng/ml 8-MOP and 2 joule/cm2 UVA were monitored over 5 days, and this regimen was found to be cytostatic. SMC viability was confirmed by trypan blue exclusion. CONCLUSIONS: Our studies suggest that 8-MOP/UVA photochemotherapy may represent a novel approach to the control of localized SMC proliferation.     

Alpha-tocopherol as a modulator of smooth muscle cell proliferation

The effects of alpha-tocopherol and beta-tocopherol have been studied in rat and human aortic smooth muscle cells. Alpha-tocopherol, but not beta-tocopherol, inhibited smooth muscle cell proliferation and protein kinase C in a dose-dependent manner, at concentrations ranging from 10 to 50 microM. Beta-tocopherol added simultaneously with alpha-tocopherol prevented both proliferation and protein kinase C inhibition. Protein kinase C inhibition was cell cycle-dependent and it was prevented by okadaic acid, a protein phosphatase inhibitor. Protein kinase C activity measured from aortas of cholesterol-fed rabbits was also inhibited by alpha-tocopherol. By using protein kinase C (PKC) isoform-specific inhibitors and immunoprecipitation reactions it was found that PKC-alpha was selectively inhibited by alpha-tocopherol. Further, an activation of protein phosphatase 2A by alpha-tocopherol was found, which caused PKC-alpha dephosphorylation and inhibition. Ultimately, this cascade of events at the level of cell signal transduction leads to the inhibition of smooth muscle cell proliferation.     

Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.     

Ultrastructure, calcium accumulation, and contractile response in smooth muscle

After removing extracellular Ca2+ with [ethylene bis(oxyethylenenitrilo)]tetraacetic acid, we found that the guinea pig vas deferens (VD) was mechanically responsive to electrical stimulation for a significantly greater length of time than was guinea pig taenia coli (TC). An obvious explanation for these findings is that the VD has more intracellular calcium available for contraction than does the TC. To determine if this explanation is plausible, the volume of internal storage structures within the two muscles was compared. It was found that the volumes of potential sequestering structures in the VD and TC are not significantly different. Next, the affinities of the storage structures for calcium were compared. The VD was found to accumulate approximately twice as much 45Ca as did the TC, as determined by 45Ca autoradiography. Calcium-45 was present to a greater extent in association with surface vesicles, sarcoplasmic reticulum (SR), and mitochondria than in the unassociated state within the cytoplasmic matrix. Based on the results of these experiments, we suggest that the VD and the TC of the guinea pig differ in the affinity of their storage sites for calcium.     

Dipyridamole inhibits PDGF- and bFGF-induced vascular smooth muscle cell proliferation

Dipyridamole is the only pharmacologic agent demonstrated to reduce polytetrafluoroethylene (PTFE) graft occlusion in hemodialysis patients. However, the mechanism of action of dipyridamole in preventing graft occlusion is unknown. The purpose of this study was to examine the direct effects of dipyridamole on both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation. Human aortic smooth muscle cells were grown to confluence in 96 well plates. A total of 5 x 10(-6) molar dipyridamole, PDGF 10 ng/ml, or bFGF 10 ng/ml were added to appropriate wells at the start of each experiment. Cell proliferation at 48 hours was determined using tritiated thymidine uptake. Intracellular cyclic AMP (cAMP) was measured using a competitive enzyme immunoassay. Treatment of VSMC with 5 microM dipyridamole dramatically reduced basal proliferation rates compared to controls [5229 +/- 1131 counts per minute (CPM) versus 387 +/- 68 CPM, P < 0.001]. Treatment with dipyridamole also reduced PDGF-stimulated VSMC proliferation (7311 +/- 1655 CPM vs. 593 +/- 110 CPM, P < 0.001) as well as the response to bFGF (5632 +/- 1270 CPM vs. 310 +/- 31 CPM, P < 0.001). Treatment of VSMC with either 5 or 20 microM dipyridamole did not change intracellular cAMP levels. Furthermore, the addition of dibutyryl cAMP to VSMC demonstrated only a modest inhibitory effect on proliferation. We conclude that dipyridamole inhibits both PDGF- and bFGF-stimulated VSMC proliferation. The effects of dipyridamole on VSMC proliferation do not appear to be entirely mediated by changes in intracellular cAMP concentrations. The direct effect of dipyridamole on VSMC proliferation may account for its efficacy in reducing PTFE graft thrombosis in hemodialysis patients.     

Rectification in the smooth muscle cell membrane of rabbit aorta

1. The current-voltage relation of the smooth muscle cell membrane of rabbit aorta was determined by the partition method. 2. No anomalous rectification was observed in any of the following solutions: normal Krebs, Na free choline, Na sulphate, and high K-Na free sulphate. 3. Delayed rectification was seen on application of depolarizing current in both normal Krebs solution and Na free choline solution. 4. High concentration of K made the steady-state current-voltage relation almost linear in a voltage range of about 0 to -20mV. This effect, and steady-state cathodal rectification which was seen in physiological solution, could be explained qualitatively by constant field theory without involving channels capable of anomalous rectification. 5. A slow decrease in K conductance, during application of large and long-lasting hyperpolarizing currents, which occurs in skeletal muscle and is attributed to the tubule system, was never observed in the arteries either in Krebs, Na-free choline, or Na sulphate solution.