Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

Homologous calcium-binding proteins in the activation of skeletal, cardiac, and smooth muscle myosin light chain kinases

In order to identify the physiological regulator of calcium dependent myosin light chain kinases of cardiac, skeletal, and smooth muscles, the effects of the three homologous calciproteins, calmodulin, troponin C, and parvalbumin, on the kinases isolated from bovine myocardium, rabbit skeletal muscle, and turkey gizzard were examined. Only calmodulin was effective in stimulating the cardiac, skeletal, or smooth muscle kinase; troponin C and parvalbumin exhibited no activation of any of the three kinases, even when examined at concentrations as high as 10-(5) M. It is concluded that calmodulin is the specific regulator of myosin light chain kinase in cardiac, skeletal, and smooth muscle.     

Dependence of IL-4, IL-13, and nematode-induced alterations in murine small intestinal smooth muscle contractility on Stat6 and enteric nerves

IL-4 and IL-13 promote gastrointestinal worm expulsion in part through effects on nonlymphoid cells, such as intestinal smooth muscle cells. The roles of Stat6 in IL-4-, IL-13-, and parasitic nematode-induced effects on small intestinal smooth muscle contractility were investigated in BALB/c wild-type and Stat6-deficient mice treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or IL-13 for 7 days. Separate groups of BALB/c mice were infected with Nippostrongylus brasiliensis or were drug-cured of an initial Heligmosomoides polygyrus infection and later reinfected. Infected mice were studied 9 and 12 days after inoculation, respectively. Segments of jejunum were suspended in an organ bath, and responses to nerve stimulation and to acetylcholine and substance P in the presence and absence of tetradotoxin, a neurotoxin, were determined. Both IL-4 and IL-13 increased smooth muscle responses to nerve stimulation in wild-type mice, but the effects were greater in IL-13-treated mice and were absent in IL-13-treated Stat6-deficient mice. Similarly, hypercontractile responses to nerve stimulation in H. polygyrus- and N. brasiliensis-infected mice were dependent in part on Stat6. IL-13, H. polygyrus, and N. brasiliensis, but not IL-4, also increased contractility to acetylcholine by mechanisms that involved Stat6 and enteric nerves. These studies demonstrate that both IL-4 and IL-13 promote intestinal smooth muscle contractility, but by different mechanisms. Differences in these effects correlate with differences in the relative importance of these cytokines in the expulsion of enteric nematode parasites.     

The effects of methylxanthines, ethymizol, ephedrine and papaverine on guinea pig and dog trachea

The study was aimed to compare the effects of pentoxyphylline, aminophylline, choline theophyllinate and ethymizol on guinea pig and dog trachea with those of theophylline, papaverine and ephedrine. The effects of these drugs on the basal tension, on dose-response curves for muscle contraction produced by histamine and on cAMP level were investigated in guinea pig trachea, together with their influence on the resting and histamine-evoked mechanical and membrane activities of dog trachea. Like papaverine, pentoxyphylline did not alter the resting membrane potential, although it relaxed both tracheal preparations, and it antagonised the effects histamine and raised the cAMP level of the smooth muscle. The effects of ethymizol were similar to those of theophylline and its water soluble derivatives (aminophylline and choline theophyllinate). Whereas, ephedrine although it decreased the basal tension and inhibited histamine-evoked responses, also elicited substantial hyperpolarization of the smooth muscle membrane with no effect on the cAMP level. These findings are consistent with the hypothesis that cAMP has an important role in the action of some bronchodilator drugs; however, it is concluded that the possibility of contributing of their action on membrane potential to their action needs to be considered. The similarity of the potencies of ethymizol and pentoxyphylline to that of classical bronchodilators in inhibiting contraction of guinea pig and dog tracheal smooth muscle suggests that they may have a therapeutic value.     

Effects of different injurious stimuli on cell death, proliferation, and collagen secretion by rabbit aortic smooth muscle cells and human umbilical vein endothelial cells in culture

Rabbit aortic smooth muscle cells were exposed to hypoxia and the DMSO-soluble particulate matter of cigarette smoke (DSP) and the effects on the cells were compared to those of ultraviolet (UV) light, a wellknown cytotoxic stimulus. Hypoxia reduced cell proliferation and collagen secretion as long as it lasted (1, 3 and 24 hours), but these effects were reversible and no persistent toxic effects were seen for the next 48 hours, measured as leakage of prelabelled [3H]-thymidine from cells. DSP caused similar changes to those produced by UV-light. Proliferation and collagen secretion were reduced, and these effects were not reversible when the stimulus was removed after one hour. DSP was toxic as there was an increased leakage of [3H]-thymidine from cells. Increasing the concentration of DSP caused more pronounced effects while extending the incubation time to 3 hours did not, the latter being in contrast to the consequences of UV-irradiation. Endothelial cells were more sensitive to the effects of DSP than smooth muscle cells. DSP diluted 1:25 compared to the smallest concentration that had significant effects on smooth muscle cells reduced proliferation of endothelial cells, and in dilutions of 1:4 almost all of the cells detached. A possible role for DSP in the development of atherosclerosis is discussed.     

Heterogeneity of desmin, the muscle-type intermediate filament protein, in blood vessels and astrocytes

Monoclonal antibodies were isolated from mice immunized with chicken gizzard desmin. Antibodies reacting with desmin on immunoblots and selectively decorating chicken and rat intestinal smooth muscle as well as the Z-line in striated muscle, were selected for this study. Based on their staining pattern on cryostat sections of chicken and rat cerebellum, spleen, kidney, aorta and femoral artery, monoclonal supernatants could be divided in three groups: (i) antibodies decorating astrocytes and vascular smooth muscle; (ii) antibodies decorating only vascular smooth muscle; (iii) antibodies decorating only astrocytes. Antibodies in group (i) and (iii) also stained GFA-negative Bergmann glia in chicken cerebellum. It is proposed that desmin may vary depending on the histological localization.     

Direct inhibitory effect of adrenomedullin, calcitonin gene-related peptide, calcitonin, and amylin on cholecystokinin-induced contraction of guinea-pig isolated caecal circular smooth muscle cells

We recently reported the direct inhibitory effect of adrenomedullin on caecal circular smooth muscle cells via cAMP system. This study was designed to determine whether the structurally related peptides to adrenomedullin (i.e.; calcitonin gene-related peptide (CGRP), calcitonin, and amylin) can inhibit the cholecystokinin octapeptide (CCK-8)-induced contractile response by exerting a direct action on guinea-pig caecal circular smooth muscle cells, and to compare the inhibitory potency of these peptides. In addition, to elucidate each intracellular mechanisms, the effects of an inhibitor of cAMP-dependent protein kinase, inhibitors of particulate or soluble guanylate cyclase on the each peptide-induced relaxation were investigated. Adrenomedullin, CGRP, calcitonin, and amylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.14 nM, 0.37 nM, 5.4 nM, and 160 nM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by all of these peptides. On the contrary, inhibitors of particulate or soluble guanylate cyclase did not have any significant effect on the relaxation produced by these peptides. In this study, we demonstrated the direct inhibitory effects of the structurally related peptides to adrenomedullin (i.e.; CGRP, calcitonin, and amylin) on the isolated caecal circular smooth muscle cells via cAMP system. The order of potency was as follows; adrenomedullin falling dots CGRP > calcitonin > amylin.     

Effects of 5-Fluorouracil in Nuclear and Cellular Morphology,Proliferation, Cell Cycle,Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.     

Effects of active substances fromStaphylococcus aureus (protein a and peptidoglycan) on neurotransmitter-induced contractions of smooth muscles

Modulatory effects of protein A (PA) and peptidoglycan (PG) fromStaphylococcus aureus on the smooth muscle contractions elicited by neurotransmitters were studied. PA and PG were found to suppress the myometrium smooth muscle contractions elicited by acetylcholine applications. The effects of the substances on the contractions were due to non-competitive inhibition through an enhancement of the potassium-dependent conductance of the smooth muscle cell membrane. PA and PG did not affect the myometrium contractions elicited by oxytocin, or the contractions of the coronary arteries elicited by acetylcholine or noradrenaline.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 30–35, January–February, 1996.     

Dietary Selenium Influences Calcium Release and Activation of MLCK in Uterine Smooth Muscle of Rats

We sought to elucidate the effects of different concentrations of dietary selenium on calcium ion release, MLCK levels, and muscle contraction in the uterine smooth muscle of rats. The selenium (Se) content of blood and of uterine smooth muscle tissues was detected by fluorescence spectrophotometry. Ca²⁺ content was measured by atomic absorption spectroscopy. Calmodulin (CaM) and MLCK RNA and protein levels were analyzed by quantitative real-time polymerase chain reaction and Western blot, respectively. Dietary Se intake increased the Se levels in the blood and in uterine smooth muscle tissues and increased the Ca²⁺ concentration in uterine smooth muscle tissues. The addition of Se also promoted CaM expression and enhanced MLCK activation in uterine smooth muscle tissues. In conclusion, Ca²⁺, CaM, and MLCK were regulated by Se in uterine smooth muscle; Se plays a major role in regulating smooth muscle contraction in the uterus.     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.     

Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture

Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture. However, smooth muscle cells change their phenotype when placed in culture. The objective of the present study was to investigate the effects of angiotensin II on (3)H-thymidine and (3)H-proline incorporation in vascular smooth muscle cells in culture and in the tunica media of blood vessels perfused at normal physiological pressures in organ culture, thus avoiding the phenotypic changes observed in cell culture. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where thoracic rat aortae were placed. Angiotensin II induced an increase in (3)H-thymidine and (3)H-proline incorporation in both culture systems. The results suggest that angiotensin II may play a role in mediating cell growth in vascular smooth muscle cells in their 'contractile' as well as in their 'synthetic' phenotype.     

Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries

To assess effects of smooth muscle energy state and intracellular pH (pH(i)) on pulmonary arterial tone during hypoxia, we measured ATP, phosphocreatine, P(i), and pH(i) by (31)P-NMR spectroscopy and isometric tension in phenylephrine-contracted rings of porcine proximal intrapulmonary arteries. Hypoxia caused early transient contraction followed by relaxation and late sustained contraction. Energy state and pH(i) decreased during relaxation and recovered toward control values during late contraction. Femoral arterial rings had higher energy state and lower pH(i) under baseline conditions and did not exhibit late contraction or recovery of energy state and pH(i) during hypoxia. In pulmonary arteries, glucose-free conditions abolished late hypoxic contraction and recovery of energy state and pH(i), but endothelial denudation abolished only late hypoxic contraction. NaCN had little effect at 0. 1 and 1.0 mM but caused marked vasorelaxation and decreases in energy state and pH(i) at 10 mM. These results suggest that 1) regulation of tone, energy state, and pH(i) differed markedly in pulmonary and femoral arterial smooth muscle, 2) hypoxic relaxation was mediated by decreased energy state or pH(i) due to hypoxic inhibition of oxidative phosphorylation, 3) recovery of energy state and pH(i) in hypoxic pulmonary arteries was due to accelerated glycolysis mediated by mechanisms intrinsic to smooth muscle, and 4) late hypoxic contraction in pulmonary arteries was mediated by endothelial factors that required hypoxic recovery of energy state and pH(i) for transduction in smooth muscle or extracellular glucose for production and release by endothelium.     

Thromboxane A2 fails to induce proliferation of smooth muscle cells enriched with eicosapentaenoic acid and docosahexaenoic acid.

Thromboxane A2 (TXA2) released from aggregating platelets and injured vessel wall stimulates smooth muscle cell proliferation, which may contribute to the development of vascular lesion formation after percutaneous transluminal coronary angioplasty. Polyunsaturated fatty acids (n-3) present in the fish oils have been shown to have anti-atherosclerotic effects. In view of this, we examined the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the active ingredients of fish oils on TXA2 induced smooth muscle cell proliferation. To find out the specificity of these fatty acids we used gamma-linolenic acid (n-6) and oleic acid (n-9) as controls. It was found that TXA2 failed to stimulate proliferation of smooth muscle cells preloaded with EPA or DHA but not with gamma-linolenic acid or oleic acid. Further, when smooth muscle cells were preloaded with both EPA and DHA, they acted together in preventing the TXA2 induced smooth muscle cell proliferation. These results demonstrate that one of the mechanisms by which fish oils may prevent neointima formation is by making smooth muscle cells less responsive to TXA2 induced proliferation of smooth muscle cells.     

Effects of a Component of Green Tea on the Proliferation of Vascular Smooth Muscle Cells

The effects of a component of green tea on the proliferation of smooth muscle cells were measured in terms of [³H]thymidine uptake. When green tea tannin mixture was added to the medium of cultured smooth muscle cells, it suppressed the proliferation of the cells dose-dependently. Similarly to the effects of the green tea tannin mixture, (–)-epigallocatechin 3-O-gallate, its main ingredient, had an inhibitory effect on smooth muscle cell proliferation at a low concentration. (–)-Epicatechin 3-O-gallate was also an effective component. Among four types of gallate-free tannin, (–)-epigallocatechin, (–)-epicatechin, and (+)-catechin showed significant dose-dependent inhibition of smooth muscle cell proliferation. However, caffeine and theanine were found to have no such action.     

Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.     

Gender, sex hormones, and vascular tone

The greater incidence of hypertension and coronary artery disease in men and postmenopausal women compared with premenopausal women has been related, in part, to gender differences in vascular tone and possible vascular protective effects of the female sex hormones estrogen and progesterone. However, vascular effects of the male sex hormone testosterone have also been suggested. Estrogen, progesterone, and testosterone receptors have been identified in blood vessels of human and other mammals and have been localized in the plasmalemma, cytosol, and nuclear compartments of various vascular cells, including the endothelium and the smooth muscle. The interaction of sex hormones with cytosolic/nuclear receptors triggers long-term genomic effects that could stimulate endothelial cell growth while inhibiting smooth muscle proliferation. Activation of plasmalemmal sex hormone receptors may trigger acute nongenomic responses that could stimulate endothelium-dependent mechanisms of vascular relaxation such as the nitric oxide-cGMP, prostacyclin-cAMP, and hyperpolarization pathways. Additional endothelium-independent effects of sex hormones may involve inhibition of the signaling mechanisms of vascular smooth muscle contraction such as intracellular Ca2+ concentration and protein kinase C. The sex hormone-induced stimulation of the endothelium-dependent mechanisms of vascular relaxation and inhibition of the mechanisms of vascular smooth muscle contraction may contribute to the gender differences in vascular tone and may represent potential beneficial vascular effects of hormone replacement therapy during natural and surgically induced deficiencies of gonadal hormones.