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Electromagnetic Fields↗
Biomedical subjects
D Leszczynski
Publications and source records attributed to D Leszczynski.
Mast cell chymase induces apoptosis of vascular smooth muscle cells.
In human coronary atheromas, the numbers of degranulated mast cells and of apoptotic smooth muscle cells (SMCs) are increased. Accordingly, the possibility exists that mast cells participate in the regulation of SMC apoptosis in the lesions. Mast cells isolated from the serosal cavities of rats were stimulated to release their secretory granules. The neutral protease chymase, present in the exocytosed granules, was found to induce apoptosis when added to rat aortic SMCs in culture. The chymase-induced apoptosis of SMCs was detected by flow cytometry, microscopic analysis of cellular morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and electrophoretic demonstration of DNA laddering. Chymase induced SMC apoptosis in a dose- and time- dependent manner, and its proteolytic activity was essential for the proapoptotic effect. In addition to rat chymase, recombinant human chymase was also found to induce apoptosis of human coronary artery SMCs in culture. These results suggest that mast cells may participate in the apoptotic regulation of SMCs in atherosclerotic lesions.
Animals↗
Laser-beam-triggered microcavitation: a novel method for selective cell destruction.
We describe a new method of cell destruction that may have potential for use in antitumor therapy. Cells are loaded by phagocytosis with microparticles (<1 microm) and irradiated with short laser pulses. Absorption of laser energy by the microparticles causes localized vaporization of the fluid surrounding the microparticles, leading to the generation of transient vapor bubbles (microcavitation) around the microparticles. Using cultures of bovine aortic endothelial cells, we demonstrate that induction of intralysosomal microcavitation is an efficient, rapid and selective method of cell killing that is dependent on the number of microparticles, the number of laser pulses, and the fluence of the laser pulses. Cell killing by microcavitation is a very selective process that is restricted to cells containing microparticles, leaving other cells unaffected. Intracytoplasmic release of lysosomal hydrolases is, in part, responsible for cell death, because the protease inhibitors E64d and TLCK diminished cell killing. Using the broad-specificity caspase inhibitor Z-VAD-fmk, we determined that lysosomal hydrolases could induce apoptosis in a caspase-independent manner. We also examined the possibility of microcavitation-induced delayed effects in the cells that survived the treatment. Using flow cytometry, we determined that there was no delayed cell death between 1 and 4 days after microcavitation. Moreover, we did not observe changes in the cell cycle, in expression of the proteins BCL2, HSP70 and HSP27, or in PARP degradation. In conclusion, microcavitation induces rapid and specific cells death (limited only to cells containing microparticles), without producing delayed effects among the surviving cells.
Animals↗
Photodynamic therapy generates a matrix barrier to invasive vascular cell migration.
Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm(2); photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 microg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (P<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (P<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.
Animals↗
Photodynamic therapy induces apoptosis in intimal hyperplastic arteries.
Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light (lambda = 675 nm) 100 J/cm(2) was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an alpha-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.
Actins↗
Association of p53, K-ras and proliferating cell nuclear antigen with rat lung lesions following exposure to simulated nuclear fuel particles.
Expression of p53, K-ras, and proliferating cell nuclear antigen (PCNA) and mutations of p53 and K-ras genes in lung lesions of Han/Wistar rats were investigated by immunohistochemistry and direct DNA sequencing following a long-term exposure of animals to neutron-activated UO2 particles. The p53 protein was overexpressed in all five malignant tumors, in 62% of benign tumors, and in 42% of hyperplastic lesions examined. K-ras protein and PCNA levels were only slightly elevated in all types of lung lesions. In three malignant tumors a C-->T transition was detected in codon 288 (human 290) of the p53 gene, but this mutation was not present in seven other tumors analyzed. No mutations were detected in codons 12/13 and 61 of the K-ras gene in any of the five tumors analyzed. Our findings suggest that K-ras overexpression is a rare alteration, whereas p53 protein overexpression (sometimes associated with mutated p53 gene), as assessed with the CM5 antibody, is a relatively common phenomenon in hot particle-induced preneoplastic and neoplastic rat lung lesions.
Animals↗
Different effects of photodynamic therapy and gamma-irradiation on vascular smooth muscle cells and matrix : implications for inhibiting restenosis.
gamma-Irradiation (gamma-RT) and photodynamic therapy (PDT) are known to inhibit intimal hyperplasia. The common mechanism is that both modalities produce free radicals, but unlike gamma-RT, PDT generates them through the absorption of light by photosensitizers. The purpose of this in vitro study was to assess the differences that PDT and gamma-RT have on the fibroproliferative response after vascular injury by comparing their effects on vascular smooth muscle cells (SMCs) and on the extracellular matrix (ECM). Mitochondrial activity (tetrazolium salt), proliferation ([(3)H]thymidine incorporation), and the mechanisms of cell death (terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling [TUNEL] staining) were used to assess differences between PDT (100 J/cm(2)) and gamma-RT (10 or 20 Gy) on SMC injury. The different effects on bioregulatory molecules were investigated by quantitating the proliferation of SMCs cultured with conditioned medium and on treated ECM. PDT of SMCs reduced proliferation and mitochondrial activity (0.5+/-0.75% and 1.7+/-4.25%, respectively, P<0.0001), whereas gamma-RT of SMCs decreased cell proliferation but did not affect metabolic activity. Stimulation with calf serum of gamma-RT-treated SMCs did not affect proliferation but increased mitochondrial enzyme activity (160+/-11%, P<0.0005). The conditioned medium, derived from PDT- but not gamma-RT-treated SMCs, did not stimulate effector SMC proliferation compared with gamma-RT-treated SMCs (16+/-4.1% versus 80+/-16.8%, P<0.0001). Apoptosis was the principle cytotoxic mechanism after PDT, whereas gamma-RT cells were growth arrested but viable. PDT of the ECM reduced effector SMC proliferation compared with controls and gamma-RT cells (18+/-6.5% versus 100+/-17.7% and 84+/-8.9%, respectively, P<0.0001). These data suggest that gamma-RT and PDT may inhibit restenosis but by different mechanisms. The effects of PDT are more diverse and may result in improved outcome while avoiding the teratogenic exposure due to ionizing irradiation.
Animals↗
Protein kinase C-alpha regulates proliferation but not apoptosis in rat coronary vascular smooth muscle cells.
In a previous study (Am. J. Pathol. 1994, 145: 1265-1270) we found rat coronary vascular smooth muscle cell (SMC) proliferation and apoptosis to be regulated by protein kinase C (PKC). In the present study we analysed whether selective depletion of alpha isozyme of PKC would affect SMC proliferation and/or apoptosis. First, using Western blot technique, it was determined that the rat SMC express alpha, delta, epsilon and zeta isozymes of PKC. The selective depletion of PKC-alpha in SMC was achieved by exposing cells to antisense oligodeoxynucleotide to mRNA for PKC-alpha (AS-PKC-alpha). The effect of AS-PKC-alpha on SMC proliferation was analysed by measurement of 3H-thymidine incorporation. The results indicated that a single dose of AS-PKC-alpha at a concentration of 10-100microM caused long-lasting (for at least 4 days) inhibition (up to 55%) of 3H-thymidine incorporation by SMC. This observation indirectly demonstrates that PKC-alpha regulates SMC proliferation. However, it was not possible to induce a significant level of apoptosis in SMC exposed even to the highest dose of AS-PKC-alpha. These data, in conjunction with the previously shown induction of apoptosis in SMC by calphostin C, suggests that another isozyme of PKC is likely to be involved in regulation of SMC apoptosis. Finally, we observed that induction of apoptosis via PKC-dependent mechanism is prevented by supplementing the culture medium with serum. This shows striking similarity with the regulation of apoptosis by the c-myc-dependent pathway. In conclusion, PKC-alpha joins the group of proteins such as c-myc, proliferating-cell nuclear antigen and cdc2 kinase which may be therapeutical targets, for antisense oligodeoxynucleotides, in order to prevent SMC hyperplasia.
Animals↗
The effects of the broadband UVA radiation on myeloid leukemia cells: the possible role of protein kinase C in mediation of UVA-induced effects.
We examined the effects of broadband UVA radiation (320-400 nm) on a rat myeloid leukemia cell line-chloroma (ChL). A Phillips face tanner model HB 171/A was used as a light source. Chloroma were irradiated through a 5 mm thick glass filter that cut off all of the UVB contamination. The irradiances were measured, from 250 to 400 nm, with a well-characterized and calibrated double-grating spectroradiometer Optronic 742. The overall uncertainty of dose evaluation was estimated to be +/-15% (2 sigma). The cells were irradiated with UVA doses of 4 and 8 J/cm2 and cultured thereafter for 24 h. After this period of time, a marked decline up to 50% was observed in cell proliferation in UVA-irradiated ChL cultures. The cell proliferation decline was found to be caused by simultaneously occurring G2/M phase cell cycle arrest and apoptosis in part of the UVA-irradiated ChL population. Concomitantly, with the decline in cell proliferation, an increase was observed in the expression of the major histocompatibility (MHC) class I and II antigens. Because protein kinase C (PKC) is known to regulate cell proliferation, apoptosis and expression of MHC antigens, and because UVA was shown to regulate PKC activity/expression, we therefore examined whether UVA irradiation has any effect on the expression of isozymes of PKC. Western blots revealed that ChL express alpha, beta I, delta, epsilon, eta, and zeta/iota isozymes of PKC and that expression of all isozymes declined 24 h after UVA irradiation (8 J/cm2). Finally, PKC activation in ChL by exposure to phorbol ester caused cell cycle arrest in G1 phase but did not induce apoptosis. This suggests that the previously shown UVA-induced PKC activation in ChL might be responsible for the induction of MHC antigens but the simultaneously observed ChL apoptosis is likely to be mediated by PKC down-regulation. All together, our results suggest that UVA, at irradiance levels that resemble the outdoor exposure, may have profound effects on the immune-related properties of leukocytes. Thus, we speculate that in vivo the immune functions of leukocytes passing through dermal capillaries might be altered by exposure to solar UVA radiation.
Animals↗
Angiopeptin, a somatostatin-14 analogue, decreases adhesiveness of rat leukocytes to unstimulated and IL-1 beta-activated rat heart endothelial cells.
We have previously demonstrated that somatostatin-14 and its octapeptide analogue, angiopeptin, decrease the ability of rat heart endothelial cells to bind leukocytes [Leszczynski, et al., Reg. Pept. 43 (1993) 131-140]. Here, we examined whether exposure of leukocytes to angiopeptin modifies their adhesiveness to the unstimulated and to IL-1 beta-activated endothelium. Monolayers of unstimulated endothelial cells bind 274 +/- 12 leukocytes/mm2. Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 microM) reduced significantly (p < 0.05) adhesion of leukocytes from 274 +/- 12 to 188 +/- 10, 185 +/- 8 and 172 +/- 3 cells/mm2, respectively. Stimulation of endothelial cells with Il-1 beta (100 U/ml) for 24 hours increased endothelial adhesiveness from 274 +/- 12 to 381 +/- 17 adhering leukocytes/mm2. Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 microM) reduced significantly (p < 0.05) binding of leukocytes to IL-1 beta-activated endothelium from 381 +/- 17 to 237 +/- 8, 254 +/- 11 and 248 +/- 13 cells/mm2, respectively. Angiopeptin had no effect on the expression of lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) by leukocytes, as assessed by flow cytometry. This suggests that angiopeptin modulates adhesive properties of leukocytes by (1) altering the expression of other than LFA-1 adhesion molecule(s) and/or (2) modulating the affinity of adhesion molecule(s) expressed by leukocytes. In conclusion, our results demonstrate that angiopeptin reduces leukocyte adhesiveness to unstimulated and to IL-1 beta-activated endothelium. It suggests that angiopeptin may suppress immune response via modulation of the leukocyte-endothelial interaction.
Animals↗
Long-wave ultraviolet radiation causes increase of membrane-bound fraction of protein kinase C in rat myeloid leukemia cells.
We examined the effect of long-wave ultraviolet radiation (UVA) on protein kinase C (PKC) and on the proliferation of rat myeloid leukemia cell line (ChL). Exposure of cells to a single dose of UVA (8 J/cm2 at 372 +/- 10 nm) caused a rapid increase in the quantity of the membrane-bound PKC, as assessed by 3H-phorbol ester (3H-PMA) binding assay (performed at 4 degrees C). Within 2 h of UVA irradiation, three peaks of increased 3H-PMA binding to the ChL cells (by 70-100%) were observed at ca. 20, 60 and 95 min post-irradiation. The exposure of ChL to UVA caused also a rapid, but transient, decline in the cell proliferation rate (by 18% within 24 h). However, the statistically significant decrease in cell numbers was observed only 3 days later (down by 22%). The inhibition of ChL proliferation was not due to alteration of cell viability as determined by trypan blue exclusion assay, and neither was it caused by cell cycle arrest or apoptosis, as determined by flow cytometry analysis of propidium iodide-labelled cells and cell morphology in May-Grünvald-Giemsa-stained cell smears. Phorbol-ester-induced activation of PKC (performed at 37 degrees C) caused inhibition of ChL proliferation similar to that caused by UVA. This suggests that a UVA-induced increase of the membrane-bound fraction of PKC may be responsible for the UVA-induced inhibition of ChL proliferation.
Animals↗
Regulation of cell cycle and apoptosis by protein kinase C in rat myeloid leukemia cell line.
The role of protein kinase C (PKC) in regulation of the cell cycle and induction of apoptosis was examined in a rat myeloid leukemia cell line--chloroma. Exposure of chloroma cells to calphostin C (a specific PKC inhibitor) led to inhibition of cell proliferation, caused by (i) partial and transient arrest of cells in the G1 phase of the cell cycle and (ii) induction of apoptosis in part of the cell population. The calphostin-C-induced inhibition of PKC activity was accompanied by changes in the expression of proliferating cell nuclear antigen (PCNA)--a protein known to participate in regulation of DNA replication and repair. Flow cytometry and western blot analyses of the PCNA expression showed that, following the calphostin C treatment, expression of PCNA declined and attained the lowest value on day 2, followed by recovery to the control level. The decline of the PCNA expression was accompanied by changes in the molecular weight of the protein suggesting either its degradation or release of unfinished protein. The decline/recovery of the PCNA expression followed the same time-pattern as the cell cycle arrest and apoptosis. Cell apoptosis is often accompanied by cell cycle arrest, therefore, we further examined whether G1 arrest would, by itself, cause apoptosis. Exposure of the chloroma cells to various doses of hydroxyurea, a G1 arrest inducing agent, caused cell apoptosis within 24-48 h after treatment. This suggests that PKC might indirectly be responsible for the chloroma cell apoptosis induced by calphostin C. In conclusion, it appears that PKC regulates cell cycle progression by modulating the G1 to S phase transition. One of the possible targets of the PKC effects observed here might be, among others (?), PCNA--a protein involved in regulation of both cell proliferation and apoptosis.
Animals↗
IL-1 beta-stimulated leucocyte-endothelial adhesion is regulated, in part, by the cyclic-GMP-dependent signal transduction pathway.
It is well known that the exposure of endothelial cells to IL-1 beta induces an increase in endothelial cell adhesiveness for leucocytes. Using rat heart endothelial cells we found that exposure of endothelial cells to IL-1 beta (100 U/ml) induces a 133-fold increase in the intracellular concentration of cyclic-GMP; from 11.5 +/- 0.2 fM to 1530 +/- 117.8 fM (per 10(6) cells). Therefore, we examined whether cyclic-GMP is involved in the regulation of endothelial adhesiveness for leucocytes. Cyclic-GMP analogue, dibutyryl cyclic-GMP Methylene blue, an inhibitor of guanylate cyclaese, and KT5823, a specific inhibitor of cyclic-GMP-dependent protein kinase, inhibited both basal as well as IL-1 beta-induced endothelial cell adhesiveness for leucocytes, and KT5823 abolished the dibutyryl-cyclic-GMP-induced increase in endothelial adhesiveness. The effect of cyclic-GMP, induced by IL-1 beta treatment, on the endothelial adhesiveness may be either direct or indirect because of the time-gap between the rise in cyclic-GMP level and the increase of endothelial adhesiveness. IL-1 beta (100 U/ml) and dibutyryl-cyclic-GMP (0.01 mM) both induced an increase in the expression of intercellular adhesion molecule-1 by endothelial cells. However, the fact that KT5823 failed to prevent this increase, suggests that, although the IL-1 beta-induced increase in adhesiveness is caused by the increase in intracellular levels of cyclic-GMP, it may not be mediated through intercellular adhesion molecule-1. In conclusion, the results obtained indicate that endothelial cell adhesiveness for leucocytes is, in part, regulated by the cyclic-GMP-dependent signal transduction pathway.
Animals↗
Apoptosis of vascular smooth muscle cells. Protein kinase C and oncoprotein Bcl-2 are involved in regulation of apoptosis in non-transformed rat vascular smooth muscle cells.
We examined the effect of several inhibitors/activators of various protein kinases on the proliferation and apoptosis of nontransformed rat coronary vascular smooth muscle cells (SMC). As expected, all the compounds (calphostin C, KT5720, KT5823, verapamil, W7, and dibutyryl-cAMP) inhibited SMC proliferation, as judged by [3H]thymidine incorporation. Three (calphostin C, verapamil and dibutyryl-cAMP) of the six compounds caused occurrence of the classical apoptotic morphology in SMC. The effect of calphostin C, an inhibitor of protein kinase C, was examined in more detail due to the known involvement of this kinase in regulation of apoptosis in a variety of cell types. In SMC cultures exposed for 1, 2, and 3 days to 0.1 mumol/L calphostin C, 7 +/- 1%, 32 +/- 3%, and 29 +/- 3% of cells underwent apoptosis, respectively, as assessed by cell morphology (control cultures had 1 to 3% of apoptotic cells). The effect of calphostin C was transient in that on day 6 following exposure to this compound the number of apoptotic cells declined to control values. Simultaneous with the induction of apoptotic morphology in SMC, a decline was seen (within 24 hours) in expression of the oncoprotein Bcl-2 in morphologically nonapoptotic SMC. An altered distribution of Bcl-2 was seen in the apoptotic cells. The calphostin C-induced generation of apoptotic cells in SMC cultures and the decline/alteration of Bcl-2 expression were not accompanied by degradation of DNA into nucleosomal fragments. In conclusion, normal, nontransformed rat coronary artery vascular SMC undergo apoptosis when exposed to an inhibitor of protein kinase C (calphostin C), to a calcium channel blocker (verapamil), and to a stimulator of cAMP-dependent protein kinase (dibutyryl-cAMP). The induction of apoptosis by the inhibitor of protein kinase C is accompanied by alterations in the Bcl-2 expression but not by DNA fragmentation.
Animals↗
Angiopeptin, the octapeptide analogue of somatostatin, decreases rat heart endothelial cell adhesiveness for mononuclear cells.
The effect of angiopeptin, a stable analogue of somatostatin, was studied on basal and interleukin-1-beta-induced endothelial cell adhesiveness for mononuclear cells, and compared to the effect of somatostatin. Angiopeptin and somatostatin decreased basal and interleukin-1-beta-induced endothelial cell adhesiveness for mononuclear cells. The decreased mononuclear cells adhesion to endothelial cells exposed to angiopeptin and somatostatin is not due to modulation of the expression of intrecellular adhesion molecule-1 because neither angiopeptin nor somatostatin decreased basal and interleukin-1-beta-induced expression of this adhesion molecule. The effect of angiopeptin in inhibiting endothelial cell adhesiveness for mononuclear cells was abolished by addition of dibutyryl-cyclic AMP. Angiopeptin induced a transient decrease in basal and interleukin-1-beta-induced cyclic AMP levels in endothelial cells. Exposure of unstimulated and interleukin-1-beta-activated endothelial cells to KT5720, a specific inhibitor of cyclic AMP-dependent protein kinase, decreased endothelial cell adhesiveness for mononuclear cells. Thus, angiopeptin most likely diminishes endothelial adhesiveness for mononuclear cells by affecting the cyclic AMP-dependent protein kinase signal transduction pathway. The findings suggest that angiopeptin and somatostatin may modify the development of the immune response by attenuating endothelial cell adhesiveness for mononuclear cells. Angiopeptin may have a potential clinical application as a modulator of some aspects of the immune response due to its long half-life and prolonged inhibitory effect on interleukin-1-beta induced endothelial adhesiveness for mononuclear cells.
Amino Acid Sequence↗
Rat heart smooth muscle cells express high and low affinity receptors for somatostatin-14, which are involved in regulation of cell proliferation.
We demonstrate that rat heart coronary artery smooth muscle cells express specific binding sites (receptors) for somatostatin-14. The sigmoidal shape kinetics of the somatostatin-14 binding by the cells suggests the presence of either an allosteric binding site or binding sites with different affinities towards the ligand. Scatchard analysis reveals presence of high affinity (Kd = 0.039 x 10(-9)M) and low affinity (Kd = 0.602 x 10(-9)M) binding sites. Somatostatin-14 and Angiopeptin can displace each other from the binding sites. The concentrations of Angiopeptin required to displace 28%-48% of bound somatostatin-14 (10(-9)M) are in the range of 10(-4)-10(-3)M. The concentrations of somatostatin-14 required to displace 8-27% of bound Angiopeptin (10(-6)M) are in the range of 10(-6)-10(-5)M. Thus, somatostatin-14 seems to possess much higher binding affinity than Angiopeptin. Binding of somatostatin-14 and Angiopeptin to rat smooth muscle cells triggers intracellular event(s) leading to inhibition of smooth muscle cell proliferation. Exposure of smooth muscle cells to somatostatin-14 and Angiopeptin decreases amount of phosphorylated tyrosine residues. The effect of somatostatin-14 and Angiopeptin on the expression of phosphotyrosine precedes and is most likely responsible, at least in part, for the inhibition of smooth muscle cell proliferation. This demonstrates that rat heart smooth muscle cells express physiologically active receptor(s) for somatostatin-14.
Animals↗
Direct and endothelial cell-mediated effect of cyclosporin A on the proliferation of rat smooth muscle cells in vitro.
Cyclosporin A (CsA) has been suggested to potentiate graft vascular disease by stimulation of smooth muscle cell (SMC) proliferation. Both the in vitro and in vivo data are discordant, showing both stimulatory and inhibitory effects of CsA on vascular SMC proliferation. The direct and endothelial cell-mediated effects of CsA on vascular SMC proliferation were examined in vitro using the incorporation of [3H]thymidine. All experiments were done in serum-free conditions. The exposure of SMC to CsA (0.0001 to 0.1 micrograms/ml) had no effect on proliferation. High doses of CsA (0.5 to 10.0 micrograms/ml) were toxic to the SMC and endothelial cells; 90% of SMC population died within 3 to 6 days of exposure to 10.0 micrograms/ml CsA. In the studies on the endothelial cell-mediated effect of CsA, the endothelial cell-conditioned medium (ECCM) significantly increased SMC proliferation. This stimulatory effect was significantly attenuated when the ECCM was obtained from endothelial cells exposed to CsA. Endothelin (ET) is suggested to be an endothelial-cell-derived growth factor for SMC, and implicated as a possible cause of the uncontrolled proliferation of SMC during development of graft vascular disease. Exposure of SMC to levels of recombinant ET similar to the levels found in the ECCM (0.19 + 0.01 pg/ml) significantly increased SMC proliferation. CsA increased fivefold ET concentration in the ECCM. However, despite this rise in ET levels, there was a 45% decrease in SMC proliferation. In conclusion, CsA does not exert a direct modulatory effect on SMC proliferation in vitro, but may inhibit SMC proliferation indirectly via endothelial cell-derived factors. These unidentified factor(s) inhibit SMC proliferation and abolish the mitogenic effect of ET on SMC.
Animals↗
Protein kinase C-regulated production of prostacyclin by rat endothelium is increased in the presence of lipoxin A4.
Prostacyclin is generated by cultured rat endothelial cells. Compound blocking activity of protein kinase C and cyclic nucleotide-dependent protein kinases (H7) and compound blocking interaction between Ca2+ and calmodulin (W7) diminish generation of prostacyclin in rat endothelial cells. These compounds give a synergistic effect when they are introduced to the endothelial cell cultures simultaneously. Compound HA1004, an inhibitor of cAMP- and cGMP-dependent protein kinases has no effect on prostacyclin generation. Lipoxin A4, a potent direct stimulator of protein kinase C, rapidly induces prostacyclin generation in rat endothelium in a dose- and time-dependent fashion. Lipoxin A4-induced generation of prostacyclin can be inhibited by H7 and W7 but not by HA1004. Lipoxin B4 has no significant effect on prostacyclin generation in rat endothelium. In conclusion, our results demonstrate that generation of prostacyclin by rat endothelial cells is regulated via a pathway involving protein kinase C and Ca2+.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗