The in-vitro effect of antineoplastic agents on proliferative activity and cytoskeletal components of plaque-derived smooth-muscle cells from human coronary arteries

BACKGROUND: Restenosis after successful percutaneous transluminal coronary angioplasty remains the major clinical problem limiting the long-term efficacy of the treatment. Recent advances in the understanding of the biology of restenosis indicate that its cause is predominantly a multifactorial stimulation of smooth-muscle cell proliferation. The aim of this study was to investigate the in-vitro effect of antineoplastic agents on smooth-muscle cells isolated from human coronary plaque material. METHODS: Atherosclerotic tissue from coronary arteries was extracted from 15 patients of both sexes by thrombendarterectomy. Cells were isolated using enzymatic disaggregation and identified to be smooth-muscle cells with fluorescent antibodies for smooth-muscle-specific alpha-actin. The antineoplastic agents cytarabine (500-0.005 micrograms/ml), doxorubicin (50-0.0005 micrograms/ml), and vincristine (10-0.0001 micrograms/ml) were added to the cultures. Six days after seeding, the cells were trypsinized and then counted. RESULTS: All three antineoplastic agents had a strong dose-dependent antiproliferative effect on cultured smooth-muscle cells. After the application of cytostatic agents, cells either became rounded or underwent complete lysis. Cytoskeletal elements, such as actin, microtubules, and vimentin, were largely altered. CONCLUSION: This investigation examined the potential role of antineoplastic therapy in the prevention of restenosis after coronary angioplasty. The development of new intravascular delivery systems, such as coated stents, may open the way for local antiproliferative strategies in interventional cardiology.     

Expression of smooth-muscle actin in cultured cells from human plasmacytoid myoepithelioma

BACKGROUND: Endoscopic sclerotherapy (ST), widely used as treatment of bleeding esophageal varices, might cause motility disturbances of the esophagus as well as mucosal damage. We performed this study to evaluate the long-term effects of repeated sclerotherapy on esophageal motility and mucosa. METHODS: Ten patients with liver cirrhosis and bleeding esophageal varices treated with repeated ST were evaluated after the last ST, median 52 months, by esophageal manometry and gastroscopy where forceps biopsies were taken. RESULTS: We found a significant difference in the distal esophageal sphincter intraabdominal length. The distal esophageal sphincter pressure was somewhat lower in the ST group although the difference did not reach statistical significance. There was infiltration of neutrophil leukocytes in biopsies from four patients and normal findings in the rest. CONCLUSIONS: Long-term follow-up evaluation showed statistically longer distal esophageal intraabdominal length in the ST group. No mucosal alterations were found at the histopathological investigation.     

Protein turnover in the smooth muscle cell of the aortic tunica media as visualized by radioautography

The turnover of newly formed proteins in the aortic myocyte was studied by means of quantitative light and electron microscopic radioautography. Adult and young mice were sacrificed at long intervals between 4 h and 108 days after one injection of tritiated leucine. The results showed that secretory activity, important in the post-natal myocyte, persists noticeably in the mature myocyte. The similarity in sedentary protein turnover, noted in both the thoracic and abdominal aortic segments of the adult and young animals, seems to be expressed by the same labelling patterns of the myoplasma. Turnover of exportable protein, which varies with age and anatomical level, appears to be related to the higher rate of elastin synthesis in the thoracic aorta of the young animal.     

各向同性多孔介质中Kozeny-Carman常数的分形分析

Kozeny-Carman(KC)方程是多孔介质渗流领域最著名的半经验公式,长期以来,KC方程及其推广形式被广泛用于估算多孔介质的渗透率.但是,方程中的KC常数是一个没有确切物理意义的经验常数,且被证明并非一个常数值.天然多孔介质中的孔隙分布往往表现出自相似的分形标度律.因此,根据多孔介质的分形特征利用微观几何模型计算了各向同性多孔介质的有效渗透率,并进一步推导了KC常数的解析表达式.结果表明,KC常数是由多孔介质的微结构决定的,是孔隙率和分形维数的函数,且随着孔隙率的增加而增大.     

An analysis of centriolar orientation in cultured ESK cells under the action of the calcium ionophor A23187

Addition of 20 microM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells gave an increase of Ca2+ in cytosol by more than 10 times. The maximum of [Ca2+] was achieved in 1-2 min after introduction of the drug. Later on [Ca2+] gradually decreased, and after 30 min of incubation with A23187 [Ca2+] was 3-5 times above normal level. Immunofluorescent and electron microscope studies showed no alterations in the microtubule system of interphase cells after 1-30 min treatment. The electro microscope study showed that-following 1.5 min introduction of the drug the random orientation of material and daughter centrioles changed: most of them settled down at an angle more, than 74 degrees to the substrate surface. After 3 min of A23187 treatment more than half of maternal centrioles were oriented perpendicular to the substrate surface. After 5 min of A23187 treatment, the percentage of maternal centrioles with perpendicular orientation was the same and this orientation remained for 30 min. The percentage of perpendicular daughter centrioles decreased after 3 min of treatment, and after 30 min their orientation was random. We suggest that the perpendicular orientation of centrioles to the substrate surface is mediated through centrosome-associated calcium-binding proteins.     

The effect of sodium nitroprusside on the membrane potential and mechanical tension of the smooth-muscle cells in the rat aorta

The rat aorta smooth muscles were contracted and depolarised with high potassium or phenylephrin solution which was followed by a relaxation and repolarisation after sodium nitroprusside administration. The latter effect was decreased az a combined action of phenylephrin and high potassium solution. Nitroprusside seems to act through a cGMP-dependent potassium transient activation of the smooth muscle cell membrane.     

A rare anatomic variation of the anomalous origin of all three major coronary arteries from the right sinus of Valsalva

We describe the case of a patient with angiographic evidence in the right sinus of Valsalva of anomalous origin of LCx and common origin of LAD and RCA. This anomaly, which has not been reported previously, represents a further anatomic variation of the rare anomalous origins of all coronary arteries from right sinus of Valsalva. The symptoms in our patients were related exclusively to the atherosclerotic lesions in LCx and RCA and not to the anatomic anomalies. The patient underwent a bypass graft to LCx and RCA.     

Emergency reoperations in the reconstructive surgery of abdominal aorta and major arteries of the lower extremities

The experience with treatment of 564 patients who underwent 642 reoperations on the arterial vessels, including 281 emergency operation, are presented. The causes of the development of pronounced ischemia of the lower extremities and that of the extreme situation are considered. The surgical tactics for the treatment in cases of transplant infection, erosive bleeding from aortal anastomosis etc. have been developed. The original methods for surgical correction in performance of a reoperation have been suggested, the analysis of the results of treatment has been performed.     

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.     

Single L-type calcium channels in smooth muscle cells from resistance arteries of spontaneously hypertensive rats

The amplitude of the whole-cell L-type Ca2+ channel current recorded from vascular smooth muscle cells is reportedly greater in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). However, no study has examined properties of single Ca2+ channels in arterial cells from these strains. To further test the hypothesis that activation of L-type Ca2+ channels in arterial smooth muscle cells would be enhanced in SHR, we recorded single Ca2+ channel currents in resistance mesenteric artery cells from SHR and WKY (8 to 9 weeks of age) using a cell-attached patch clamp technique. With 50 mmol/L Ba2+ in the recording pipette, the depolarizing pulse from a holding potential of -40 mV evoked the single L-type Ca2+ channel current. Opening of the single channels was more frequent in cells from SHR than from WKY. Single-channel conductance (20 pS) and open time (1 ms at 0 mV) did not differ in the two strains. The results suggest that an increased amplitude of the whole-cell current can be attributed to the enhanced opening of single Ca2+ channels in the arterial smooth muscle cells from SHR compared with WKY.     

Continuous in situ electrochemical monitoring of doxorubicin efflux from sensitive and drug-resistant cancer cells

One of the least well understood problems in cancer chemotherapy is the cross-resistance of certain tumor cells to a series of chemically unrelated drugs. Multidrug resistance (MDR) can be attributed to several different biophysical processes, among them increased drug efflux. This has been found to correlate with overexpression of the cell surface 170-kDa P-glycoprotein that actively excludes cytotoxic drugs against their concentration gradient. To better understand MDR, experimental methods are needed to study drug efflux from cancer cells. Continuous measurement of efflux of nonfluorescent drugs on the same cell culture in situ, or assessing efflux from a few cells or even a single cell, is beyond the capabilities of existing technologies. In this work, a carbon fiber (CF) microelectrode is used to monitor efflux of doxorubicin from a monolayer of two cell lines: an auxotrophic mutant of Chinese hamster ovary cells, AUXB1, and its MDR subline, CHRC5. Because doxorubicin is both fluorescent and electroactive, the results could be validated against existing data obtained optically and with other techniques on the same cell lines, with good agreement found. The electrochemical detection, however, is capable of in situ monitoring with high temporal resolution and is suitable for single-cell studies.     

High-performance liquid chromatographic method for resolving the enantiomers of mexiletine and two major metabolites isolated from microbial fermentation media

PURPOSE: This Phase I trial tests the ability of a new hyperthermia device, the transrectal ultrasound probe, to heat the prostate gland, and evaluates the toxicity of transrectal ultrasound hyperthermia (TRUSH) given with concurrent standard external beam irradiation in the treatment of locally-advanced adenocarcinoma of the prostate. METHODS AND MATERIALS: Between June, 1990 and August, 1991, 14 patients with American Urological Society Stage C2 or D1 adenocarcinoma of the prostate were treated with TRUSH concurrently with standard external beam radiotherapy to the prostate. Twenty-two heat treatments were delivered in 14 patients; 8 patients received two TRUSH procedures, each separated by 1 week. Patient age ranged between 53-86 (mean: 72) years. Three patients had well-, 6 patients had moderately-, and 5 patients had poorly-differentiated adenocarcinoma of the prostate. Karnofsky status ranged from 70-90. Standard radiotherapy to the prostate and periprostatic tissues was delivered using a four-field approach with 1.8-2 Gy daily fractions delivered 5 x/week to a total dose of 67-70 Gy calculated to the minimum tumor volume. TRUSH was delivered after transperineal placement of multipoint thermometry probes by a urologist, under transrectal ultrasound guidance. Two to three thermocouple probes containing seven sensors each were placed in the prostate in an attempt to sample temperatures throughout the gland. The sensor depth from the rectal wall ranged from 5-25 mm. RESULTS: Thirty-six percent of all sensors were heated above 42.5 degrees C averaged over 30 min; and all patients had at least some sensors within the prostate heated to temperatures > or = 42.5 degrees C. The average temperature of all sensors of all sensors (T(ave) +/- s.d.) over all treatments, however, was only 41.9 degrees C +/- 0.9 degrees C over 30 min. The maximum temperature for normal tissues outside the gland was 41.1 degrees C +/- 1.3 degrees C. Treatments have been well-tolerated with few complications. Tolerance has been "good" in 17/22, "fair" in 3/22, and "treatment limiting" in 2/22 treatments secondary to position intolerance and/or pain. There has been one episode of hypotension related to narcotic administration and three episodes of rapidly resolving pain during hyperthermia treatment. Mild hematuria has occurred in 5/22, and moderate hematuria has occurred in 2/22 transperineal thermometer catheter placements. CONCLUSION: In conclusion, TRUSH is well-tolerated and has great potential for consistently heating the prostate gland. We anticipate that further equipment modifications will improve our ability to heat the entire prostate to temperatures > 42.5 degrees C.     

Inhibitory action of insulin-sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea-pig

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.     

Role of extracellular signal-regulated kinases in angiotensin II-stimulated contraction of smooth muscle cells from human resistance arteries

BACKGROUND: We assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries. METHODS AND RESULTS: VSMCs derived from resistance arteries (<300 microm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4+/-0.36, Emax=541+/-55 nmol/L), pHi (pD2=9. 4+/-0.29, Emax=7.19+/-0.01), and contraction (pD2=9.2+/-0.21, Emax=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses. CONCLUSIONS: These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.     

Effects of nicotine on K+ channel currents in vascular smooth muscle cells from rat tail arteries

Intake of nicotine has been related in many cases to acute or chronic hypertension. Using the patch-clamp technique the effect of nicotine on voltage-dependent K+ channel currents in rat tail artery smooth muscle cells was studied. Nicotine at concentrations of 1-100 microM or 0.3-3 mM increased or decreased, respectively, the amplitude of the tetraethylammonium-sensitive K+ currents. Pretreatment of cells with 10 microM dihydro-beta-erythroidine hydrobromide, a nicotinic receptor antagonist, abolished the excitatory effect (n=6), but not the inhibitory effect (n=10), of nicotine on K+ channel currents. The activation of nicotinic receptors with 100 microM 1,1-dimethyl-4-phenylpiperazinium iodide increased K+ channel currents by 27.4+/-3.8% (n=13, P < 0.01). Our results indicate that the excitatory and inhibitory effects of nicotine on K+ channels are respectively mediated by a nicotinic receptor-dependent mechanism and by a direct interaction of nicotine with K+ channels.     

Modification of endometrial arteries during invasion by cytotrophoblast cells in the pregnant macaque

Fetal trophoblast cells invade endometrial blood vessels and gain access to maternal blood within two days after the onset of blastocyst implantation in macaques. Soon thereafter, cytotrophoblast cells migrate well into the lumina of arteries and subsequently invade arterial walls. Using electron microscopy and light microscopy we investigated the interactions between invasive cytotrophoblast cells and the cellular and extracellular components in the walls of endometrial arteries. The placentas and adjacent endometrium of 22 macaques (GD 17 to term) were examined. Spiral arteries containing migratory cytokeratin-labeled cytotrophoblast cells were identified at all stages examined. Early modification of each artery showed that a plug of intraluminal cytotrophoblast cells temporarily filled the arterial lumen in the vicinity of the trophoblastic shell. Distal to this plug the group of cells tapered as a continuous mass, filling only a portion of the lumen. Endothelial cells were displaced from their basal lamina by closely apposed cytotrophoblast cell processes. Soon thereafter these processes penetrated the basal lamina and achieved contact with smooth muscle cells of the tunica media. As cytotrophoblast cells infiltrated the arterial wall they hypertrophied and secreted extracellular matrix, thereby differentiating into intramural cytotrophoblast. The patent lumen of the artery was reestablished concomitant with the migration of intraluminal cytotrophoblast cells through the arterial tunica intima and into the tunica media. The presence of clusters of cytotrophoblast cells in the arterial wall results in discontinuity of the tunica media and dispersion of the smooth muscle. The combined changes result in expanded circumferences of invaded arteries as well as diminished ability to contract. In portions of arteries adjacent to the trophoblastic shell cytotrophoblast usually occupied the entire perimeter and thickness of the artery wall, while in areas distal only a portion of the wall was invaded. Despite extensive arterial modification, evidence of cell death among the fetal and maternal tissues involved was rare. By later gestation only a few intraluminal cytotrophoblast cells were seen. Intramural cells were surrounded by a thick layer of matrix, but maintained contact with adjacent cells through cytoplasmic processes, some of which formed gap junctions. Maternal cellular and connective tissue elements were excluded from the cytotrophoblast-matrix pads and the cytotrophoblast cells retained attributes of glycoprotein producing cells to term. Spiral arteries were modified well into the spongiosum layer of the endometrium, and some were modified into the myometrium.