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.     

Effect of flush-perfusion on vascular endothelial and smooth muscle function

BACKGROUND: The aim of this study was to investigate how much perfusion pressure an artery can tolerate without significant loss of endothelium-dependent relaxation (EDR) and vascular contractility. METHODS: The abdominal aortas of 396 Sprague-Dawley rats were used. One hundred twenty aortas were flush-perfused for 1 or 5 minutes with cold St. Thomas' Hospital cardioplegic (STHC) solution or with the same solution but modified by the addition of 3.5% dextran 40. Three perfusion pressures were tested: 50, 100, and 150 mm Hg. Two hundred eighty vessels were subjected to pressures of 50, 150, or 300 mm Hg using saline or STHC solution at 22 degrees C or STHC solution at 4 degrees C, for 10 or 60 seconds. The vessels were investigated in organ baths. Contractility was tested with the thromboxane analogue U-46619, acetylcholine was used to investigate EDR, and papaverine to elicit endothelium-independent relaxation. RESULTS: Flush-perfusion with cold STHC solution for 5 minutes at a perfusion pressure of 50 or 100 mm Hg affected neither contractility nor EDR. Vessels exposed to a flush-perfusion pressure of 150 mm Hg for 1 or 5 minutes lost 39% (p < 0.001) and 53% (p < 0.001) of their contractility, respectively. Flush-perfusion at 150 mm Hg for 1 minute did not affect EDR, whereas 5 minutes' perfusion caused a reduction of 7% (p < 0.05). A repetition of these experiments using STHC solution with 3.5% dextran 40 added gave no significantly different results. The impairment in contractility and EDR seen after perfusion at 150 mm Hg for 5 minutes disappeared after transplantation and reperfusion for 7 days. The vessels could be distended with saline or STHC solution at a pressure of 150 mm Hg without affecting contractility at 22 degrees C. At 4 degrees C, however, this pressure was harmful to contractility. Distention at a pressure of 300 mm Hg almost abolished contractility and 7 days after transplantation there had not yet been any recovery of contractility, but 30 days after transplantation the grafts had regained their normal contractility. CONCLUSIONS: Cold STHC solution, with or without dextran 40, can be used with a perfusion pressure of 100 but not 150 mm Hg without impairing EDR or vascular smooth muscle function.     

Effect of temperature in long-term preservation of vascular endothelial and smooth muscle function

BACKGROUND: In clinical transplantation the donor organ is perfused with a cold preservation solution to obtain quick core cooling and a suitable environment for the tissue cells. Without good preservation of the vasculature, progressive deterioration of the blood flow during reperfusion may ultimately lead to the no-reflow phenomenon, even though the function of the other cells in the organ may be adequately preserved. The aim of this study was to find the optimal storage temperature for preservation of the vasculature. METHODS: The infrarenal aorta of 126 Sprague-Dawley rats were studied in organ baths: as fresh controls, after 36 hours of storage at 0.5 degrees C, 4 degrees C, 8.5 degrees C, and 22 degrees C in University of Wisconsin solution, and after 36-hour storage followed by transplantation and a lapse of 2 hours, 24 hours, and 7 days. The thromboxane analogue U-46619 was used to test contractility. Acetylcholine was used to elicit endothelium-dependent relaxation (EDR), and papaverine to elicit endothelium-independent relaxation. RESULTS: Storing the vessels at 0.5 degree C proved best regarding preservation of contractility, with a nonsignificant decrease, whereas storage at 4 degrees C and 8.5 degrees C resulted in a significant decrease after 36 hours. The contractility did not recover within 24 hours of in vivo reperfusion, but full recovery was seen after 7 days. Regardless of the preservation temperature used, a significant impairment in EDR was seen after 36 hours of storage. Two hours after transplantation, vessels stored at 4 degrees C and 8.5 degrees C showed no significant impairment in EDR, whereas those stored at 0.5 degrees C demonstrated a significant loss of EDR. After 24 hours and after 7 days, EDR was normal in all groups. CONCLUSIONS: Endothelium-dependent relaxing factor function is best preserved at 4 degrees C and 8.5 degrees C, whereas preservation of vascular smooth muscle function is best preserved at 0.5 degrees C.     

Activations of mitogen-activated protein kinases and phospholipase D in A7r5 vascular smooth muscle cells

Activation of mitogen-activated protein kinases (MAPKs) was examined in the A7r5 rat vascular smooth muscle cell line. Treatment of A7r5 cells with vasopressin, phorbol ester (PMA), or serum resulted in activation of two MAPKs, Erk-1 and Erk-2. Phosphatidylinositol-specific phospholipase C was activated in response to vasopressin but not to PMA. Vasopressin and PMA both caused maximal activation of PLD within 5 minutes. Application of bacterial phospholipase D (PLD) to A7r5 cells increased phosphatidic acid to levels similar to those seen with vasopressin or PMA. Acute exposure of the cells to vasopressin, PMA, or PLD increased phosphorylation of many of the same cytosolic and membrane proteins. However, bacterial PLD did not promote significant activation of Erk-1 and Erk-2. Phosphatidic acid and lysophosphatidic acid (LPA) likewise did not stimulate MAPK activity in A7r5 cells. Serum and vasopressin stimulated DNA synthesis when present for more than 30 min, while PLD, PMA, phosphatidic acid, and LPA were not mitogenic. These data suggest that activations of MAPKs and PLD are concurrent but independent responses to vasopressin in A7r5 cells. Acute activation of these enzymes is not sufficient to simulate DNA synthesis.     

Cyclo-oxygenase-2 in vascular smooth muscle

Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.     

Sarcoplasmic reticulum-sarcolemma interactions and vascular smooth muscle tone

A characteristic of vascular smooth muscle cell morphology is a close apposition of its peripheral sarcoplasmic reticulum (SR) with the sarcolomma; this arrangement gives rise to important functional interactions whereby the peripheral SR regulates Ca2+ influx and vascular tone. We review here the key evidence supporting the following aspects of SR-sarcolemma interactions while establishing a conceptual framework encompassing (i) the SR ultrastructure and functions, (ii) the integration of the sarcolemmal Na+-Ca2+ exchanger and the peripheral SR in the mediation of a bidirectional Ca2+ exchange between the peripheral SR and the extracellular space, (iii) the existence of a higher myoplasmic free Ca2+ concentration [Ca2+]myo in the subsarcolemmal space formed between the sarcolemma and the peripheral SR relative to the [Ca2+]myo of the inner myoplasm in the resting smooth muscle cell, (iv) the division of the subsarcolemmal space into functional microdomains, (v) the existence of spontaneous localized bursts of Ca2+ release from the peripheral SR (Ca2+ sparks) towards the sarcolemma, (vi) the physiological triggering of nonlocalized Ca2+ release from the peripheral SR by Ca2+ influx (Ca2+-induced Ca2+ release), and (vii) capacitative Ca2+ entry in vascular smooth muscle. We present an overview of the physiological and pathological implications of these interactions.     

Microtubules regulate pulmonary vascular smooth muscle contraction

Two patients with cluster-tic syndrome are reported. The first, a 43-years-old man, complaining of trigeminal pain in the right side of the face, accompanied by homolateral autonomic signs, such as ocular injection, sweating and drooped eyelid. The cluster attack was triggered by chewing, shaving and washing the face. The periodicity of bouts was six months. The pain was relieved by carbamazepine (800 mg/day). The second patient, a 43-year-old man, with an excruciant, neuralgic pain in the left side of the face, accompanied by tearing, conjuntival injection, drooped eyelid, rhinorrhea, photophobia and phonophobia. The neurologic examination showed triggered points in the first and second division of the trigeminal nerve. The patient was treated with verapamil (160 mg/day) and prednisone (60 mg/day), with relief of his symptoms. The periodicity of bouts was once a year. The literature was reviewed and 37 cases previosly reported are considered. We conclude that there are two different groups of patients. In the first group, the patients had cluster and trigeminal bouts in different time. In the second group, with only nine cases, the patients presented both cluster and trigeminal type of pain at the same time, as in the two cases reported here.     

血管平滑肌细胞的增殖因素及机制

血管平滑肌细胞(VSMC)增殖已成为目前心血管疾病研究领域里的热点,VSMC增殖、迁移及凋亡在冠状动脉粥样硬化(AS)、冠状动脉搭桥术、经皮冠状动脉介入治疗(PCI)术后血管再狭窄的发生和发展过程中起重要作用.本文作者对VSMC增殖及促增殖因素和机制进行综述.     

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 actions of ketamine on vascular smooth muscle

The responses of rabbit aortic strips superfused with noradrenaline, adrenaline and 5-HT were studied alone and in combination with ketamine (50 mug/ml). Ketamine caused a slight depression of the isolated aorta but potentiated responses to adrenaline but not to noradrenaline or 5-hydroxytryptamine. Ketamine did not potentiate aortic strips contracted to a stable level by pyrogallol and adrenaline. Experiments carried out with COMT from homogenates of rat liver showed that, in contrast to pyrogallol (10(-5) M), ketamine (10(-3) M) did not inhibit the enzyme. Other experiments with rabbits given 6-hydroxydopamine showed that aortas of these rabbits responded in a similar manner to controls when treated with ketamine and catecholamines. Results obtained with aortas contracted by adrenaline and noradrenaline with ketamine present, followed by oil immersion, showed that ketamine prolonged greatly the relaxation induced by adrenaline and to a lesser extent the relaxation induced by noradrenaline. The results of these studies indicate that ketamine prevented catecholamines from reaching the intracellular site of COMT. In this respect, ketamine can be termed an inhibitor of uptake site 2. If this hypothesis is valid then the action of ketamine on vascular tissue might explain the cardiovascular effects of the drug in man and experimental animals.     

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.     

Oxidized collagen stimulates proliferation of vascular smooth muscle cells

We have examined the use of presurgical morphine-midazolam combination in 80 children aged 2-10 y undergoing repair of hypospadias. They were allocated randomly, in a double-blind study, to receive one of four morphine-midazolam combination doses (n = 20 each); (group I: 75 microg/kg each) [corrected] (group II: 75 microg/kg [corrected] morphine, 50 microg/kg [corrected] midazolam); (group III: 50 microg/kg [corrected] morphine, 75 microg/kg [corrected] midazolam); (group IV: 50 microg/kg [corrected] each). Drugs were given after induction of anesthesia and before the start of surgery. Observational scoring system, using crying, movement, agitation, posture and localization of pain as scoring criteria, was used to assess the children during their stay in the recovery room together with their sedative and/or analgesic requirement. Pre-surgical morphine-midazolam administration produced stable hemodynamic variables with satisfactory postoperative analgesia suggesting 75 microg/kg [corrected] dose of both morphine and midazolam as upper permissible dose, and 50 microg/kg [corrected] each as lower effective dose.     

Thrombin activates MAPKAP2 kinase in vascular smooth muscle

OBJECTIVE: To study the effects of estradiol and progesterone on the proliferation of normal human breast epithelial cells in vivo. DESIGN: Double-blind randomized study. SETTING: Departments of gynecology and of cell biology at a university hospital. PATIENT(S): Forty postmenopausal women with untreated menopause and documented plasma FSH levels of >30 mIU/mL and estradiol levels of <20 pg/mL. INTERVENTION(S): Daily topical application to both breasts of a gel containing a placebo, estradiol, progesterone, or a combination of estradiol and progesterone during the 14 days preceding esthetic breast surgery or excision of a benign lesion. MAIN OUTCOME MEASURE(S): Plasma and breast tissue concentrations of estradiol and progesterone. Epithelial cell cycles were evaluated in normal breast tissue by counting mitoses and performing quantitative proliferating cell nuclear antigen immunolabeling analyses. RESULT(S): Increasing the estradiol concentration enhanced the number of cycling epithelial cells, whereas increasing the progesterone concentration significantly limited the number of cycling epithelial cells. CONCLUSION(S): Exposure to progesterone for 14 days reduced the estradiol-induced proliferation of normal breast epithelial cells in vivo.     

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells

BACKGROUND: Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS: We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS: The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.     

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.     

The electrophysiological effects of tetraphenylphosphonium on vascular smooth muscle

OBJECTIVE: To investigate whether luteal secretion of inhibin-a is altered in the perimenopausal transition and to evaluate whether luteal inhibin secretion is correlated with other markers of ovarian reserve such as FSH and inhibin-b. DESIGN: Prospective study. SETTING: Reproductive Endocrinology Laboratories at The Ohio State University. PATIENT(S): Twenty-five women 39-52 years of age with regular menstrual cycles. INTERVENTION(S): Daily urine samples were monitored (LH predictor kit) to identify the day of ovulation. Blood samples obtained on days 6 and 8 after the LH surge and on day 3 of the subsequent follicular phase were assayed for FSH, E2, progesterone. inhibin-a, and inhibin-b. MAIN OUTCOME MEASURE(S): Serum levels of inhibin-a, inhibin-b, FSH, E2, and progesterone. RESULT(S): Luteal phase inhibin-a and follicular phase inhibin-b were correlated inversely with age in perimenopausal women. In addition, luteal phase inhibin-a and follicular phase inhibin-b levels were correlated inversely with follicular phase FSH levels. CONCLUSION(S): Both luteal phase inhibin-a and follicular phase inhibin-b levels are correlated inversely with age during the fifth decade of life. These findings suggest that corpus luteum function is altered during the perimenopausal transition. Moreover, these direct measures of ovarian function may be more sensitive indicators of "ovarian reserve" than indirect indicators such as pituitary FSH secretion.