The effect of miR-224 down-regulation on SW80 cell proliferation and apoptosis and weakening of ADM drug resistance.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "The effect of miR-224 down-regulation on SW80 cell proliferation and apoptosis and weakening of ADM drug resistance, by C.-Q. Liang, Y.-M. Fu, Z.-Y. Liu, B.-R. Xing, Y. Jin, J.-L. Huang, published in Eur Rev Med Pharmacol Sci 2017; 21 (21): 5008-5016-PMID: 29164556" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/13747.

[Characteristics of peripheral blood leukocyte differential counts in patients with COVID-19].

To investigate the early changes of peripheral blood leukocyte differential counts in patients with COVID-19. Ten patients with COVID-19 and 30 patients with other viral pneumonia (non-COVID-19) admitted to The Sixth People's Hospital of Shanghai and Jinshan Branch Hospital from January 22 to February 17, 2020 were enrolled in this study. The differential counts of white blood cells (WBC) were analyzed. Patients in COVID-19 group showed relatively lower absolute WBC count 4.95(3.90,6.03)×10(9)/L, lymphocyte absolute count 1.20(0.98,1.50)×10(9)/L and eosinophil absolute count 0.01(0.01,0.01)×10(9)/L. Leukopenia developed in two patients(2/10), lymphocytopenia also in two patients(2/10). Seven over ten patients presented with eosinophil cytopenia. In non-COVID-19 group, absolute WBC count was 8.20(6.78,9.03)×10(9)/L (P<0.001), lymphocyte absolute count 1.75(1.20,2.53)×10(9)/L(P=0.036), eosinophil absolute count 0.02(0.01,0.03)×10(9)/L(P=0.005). Lymphocytopenia occurred in 16.7% patients, eosinophil cytopenia in 16.7% patients too. In conclusion, leukopenia, lymphocytopenia and eosinophil cytopenia are more common in COVID-19 patients than those in non-COVID-19 patients.

[Characteristics of peripheral blood leukocyte differential counts in patients with COVID-19].

To investigate the early changes of peripheral blood leukocyte differential counts in patients with COVID-19. Ten patients with COVID-19 and 30 patients with other viral pneumonia (non-COVID-19) admitted to Shanghai Jiao Tong University Affiliated Sixth People's Hospital and Jinshan Branch Hospital from January 22 to February 17, 2020 were enrolled in this study. The differential counts of white blood cells were analyzed. Patients in COVID-19 group showed relatively lower absolute white blood cell (WBC) count 4.95(3.90,6.03)×10(9)/L, lymphocyte absolute count 1.20(0.98,1.50)×10(9)/L and eosinophil absolute count 0.01(0.01,0.01)×10(9)/L. Leukopenia developed in two patients(2/10), lymphocytopenia also in two patients(2/10). Seven over ten patients presented with eosinophil cytopenia. In non-COVID-19 group, absolute WBC count was 8.20 (6.78,9.03) ×10(9)/L (P<0.001), lymphocyte absolute count 1.75(1.20,2.53)×10(9)/L(P=0.036), eosinophil absolute count 0.02(0.01,0.03)×10(9)/L(P=0.05). Lymphocytopenia occurred in (16.7%) patients, eosinophil cytopenia in 16.7% patients too. In conclusion, leukopenia, lymphocytopenia and eosinophil cytopenia are more common in COVID-19 patients than those in non- COVID-19 patients.

The effect of miR-224 down-regulation on SW80 cell proliferation and apoptosis and weakening of ADM drug resistance.

OBJECTIVE: Glycogen synthase kinase-3ß (GSK-3ß) can phosphorylate and degrade ß-catenin, and negatively regulates Wnt/ß-catenin signal pathway. MiR-224 up-regulation is associated with colorectal cancer (CRC). Bioinformatics analysis showed complementary binding sites between miR-224 and GSK-3ß. This study investigated if miR-224 plays a role in mediating GSK-3ß expression, Wnt/ß-catenin pathway activity, CRC cell proliferation, apoptosis as well as drug sensitivity of Adriamycin (ADM). MATERIALS AND METHODS: Dual luciferase gene reporter assay demonstrated the regulatory relationship between miR-224 and GSK-3ß. Expression of miR-224, GSK-3ß, ß-catenin, and Survivin was measured in normal colon epithelium NCM460, CRC cell line SW480, and drug-resistant SW480/ADM cell line. Flow cytometry measured apoptosis under ADM with an IC50 concentration of SW480 cells, followed by CCK-8 analysis of cell proliferation. SW480/ADM cells were treated with miR-224 inhibitor and/or pSicoR-GSK-3ß, followed by analysis of the expressions of GSK-3ß, ß-catenin and Survivin, cell apoptosis, and cell proliferation by EdU staining. RESULTS: MiR-224 targeted and inhibited GSK-3ß expression. In SW480/ADM cells, GSK-3ß expression and cell apoptosis rate were lower than those in SW480 cells, whilst miR-224, ß-catenin, and Survivin expression or proliferation were higher than those in SW480 cells. Transfection of miR-224 inhibitor and/or pSicoR-GSK-3ß significantly increased GSK-3ß expression in SW480/ADM cells, and decreased ß-catenin and Survivin expression, leading to reduced proliferation potency, enhanced cell apoptosis and suppressed ADM resistance. CONCLUSIONS: MiR-224 up-regulation is associated with ADM resistance of CRC cells. Suppression of miR-224 expression up-regulated GSK-3ß expression, inhibited Wnt/ß-catenin signal pathway activity and Survivin expression, as well as reduced ADM resistance of CRC SW480 cells.

[Expression profiles of long non-coding RNAs in human liver cell line LO2 with stable expression of hepatitis B x gene].

OBJECTIVE: To investigate the differential expression profiles of long non-coding RNAs (lncRNAs) in human liver cell line LO2 with stable expression of hepatitis B x (HBx) gene, and to screen out the lncRNAs which play an important role in HBV-related liver cancer. METHODS: The lncRNA microarray was used to establish the differential expression profiles of lncRNAs, and the methods such as scatter plots and cluster analysis were used to obtain the HBx-related lncRNAs with differential expression. The qRT-PCR was used to verify some lncRNAs with differential expression. The t-test was used to compare the expression of lncRNAs between the two microarray groups, and hierarchical cluster analysis was used for the original data of lncRNAs with differential expression. RESULTS: Compared with the control group transfected with blank plasmids (L02/pcDNA3.0), LO2/HBx cells had 323 lncRNAs with > 2-fold upregulation and 421 lncRNAs whose expression was reduced by more than 50% (P < 0.05). The results of qRT-PCR verified 4 upregulated lncRNAs (TCONS_00006195, ENST00000557524, NR_037597, and ENST00000539975) and 3 downregulated lncRNAs (ENST00000508424, ENST00000447433, and uc001lva.4), which were consistent with the results of microassay. CONCLUSION: HBx-related lncRNAs are successfully screened out, which lays a foundation for further investigation of the role of lncRNAs in the pathogenesis of liver cancer.

Stability and chaotification of vibration isolation floating raft systems with time-delayed feedback control.

This paper presents a systematic study on the stability of a two-dimensional vibration isolation floating raft system with a time-delayed feedback control. Based on the generalized Sturm criterion, the critical control gain for the delay-independent stability region and critical time delays for the stability switches are derived. The critical conditions can provide a theoretical guidance of chaotification design for line spectra reduction. Numerical simulations verify the correctness of the approach. Bifurcation analyses reveal that chaotification is more likely to occur in unstable region defined by these critical conditions, and the stiffness of the floating raft and mass ratio are the sensitive parameters to reduce critical control gain.

Toluidine blue-mediated photodynamic therapy of oral wound infections in rats.

The purpose of this study was to examine the effect of toluidine blue (TB)-mediated photodynamic therapy (PDT) on oral wound infections in rats. The study called for a combination treatment of a 1mg/ml solution of TB with a red light at three intensity settings of 12 J/cm(2), 24 J/cm(2) and 48 J/cm(2). In the group that was given the highest light dose of 48 J/cm(2), an average kill rate of approximately 97% was achieved. A lesser killing effect was achieved in the group that was subjected to the lowest light dose of 12 J/cm(2), where an average of approximately 25% of the bacteria survived. After PDT, the lesions were allowed to develop, and the peak size of the lesions was larger in the control group than in the test groups, especially for the 48 J/cm(2) group. We also observed that in the 24 J/cm(2) and 48 J/cm(2) groups the lesions were of significantly smaller size. Our study demonstrated that combined TB-PDT therapy can successfully treat oral wound infections in rats. These promising results recommend the use of this treatment as a possible alternative to topical anti-microbials in future clinical applications.

Neurosteroid enhances glutamate release in rat prelimbic cortex via activation of alpha1-adrenergic and sigma1 receptors.

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of alpha1-adrenergic and sigma1 receptors, but in the hippocampus it might be dependent on sigma1 receptors only. The activation of alpha1-adrenergic receptors synergized sigma1 receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.

Congenital nasal pyriform aperture stenosis with semilobar holoprosencephaly.

We describe a child who has congenital nasal pyriform aperture stenosis with single maxillary central incisor, holoprosencephaly and central diabetes insipidus without any apparent anterior pituitary dysfunction. Conservative management of the congenital nasal pyriform aperture stenosis is adopted and management of diabetes insipidus is described. A literature review is undertaken.

Hepatotoxicity and persistent renal insufficiency after repeated supratherapeutic paracetamol ingestion in a Chinese boy.

Paracetamol has always been regarded as a useful and safe drug. The risk of toxicity with repeated supratherapeutic paracetamol is an underrecognised condition. We report on a 12-month-old boy who presented with hepatotoxicity, disseminated intravascular coagulation and persistent renal insufficiency 4 days after repeated ingestion of a supratherapeutic dosage of paracetamol. To the best of our knowledge, this is the first reported case of paediatric chronic paracetamol poisoning among the Chinese population. In addition, persistent renal insufficiency has not been a previously reported feature of chronic paracetamol poisoning. We propose that renal damage is the result of the synergistic effect of hypoperfusion and paracetamol overdose.

Decreased tissue plasminogen activator and increased plasminogen activator inhibitors and increased activator protein-1 and specific promoter 1 are associated with inhibition of invasion in human A375 melanoma deprived of tyrosine and phenylalanine.

We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) restriction significantly decreased the metastatic phenotype of the pigmented murine B16BL6 melanoma in vivo and decreased the in vitro invasion of these cells. Here we report that invasion and chemoinvasion through GFR Matrigel of the human amelanotic A375 melanoma also is significantly inhibited by Tyr and Phe deprivation in vitro. Deprivation of these two amino acids decreased the secretion and protein expression of tissue-type plasminogen activator (tPA) while expression and secretion of plasminogen activator inhibitor (PAI-1 and PAI-2) were increased. Moreover, nuclear extracts of Tyr- and Phe-deprived cells exhibited increased binding of the transcription factors, activator protein-1 (AP-1) and specific promoter-1 (Sp1), to consensus oligonucleotides as determined by electrophoretic mobility shift assay. Nuclear binding activity to the oligonucleotide consensus sequence for AP-1 was inhibited by antibody against c-Fos and more effectively inhibited by an antibody against c-Jun. We conclude that decreased invasion and chemoinvasion of A375 melanoma cells deprived of Tyr and Phe are related to decreased secretion of tPA and increased secretion of PAIs. Increased AP-1 and Sp1 binding implicates these transcription factors in the regulation of PAI expression.

Histopathological examination of chemo-sympathectomy in cats.

In recent decades, there has been an increase in both the number of sympathectomy techniques, as well as the surgical findings of sympathetic anatomy. Currently the advanced technique of C-arm guided percutaneous thoracic chemo-sympathectomy is widely used for the treatment of palmar hyperhidrosis. However, a better understanding of chemical agents in sympathectomy is required. In this study, chemo-sympathectomy was performed in cats, using alcohol, glycerol and various concentrations of phenol, to determine the chronic neurotoxic effects of these chemical agents on the stellate ganglia. The stellate ganglia of 24 cats were exposed under endotracheal general anesthesia, then injected with about 0.02 ml of absolute alcohol, glycerol and phenol (10%, 25%, 50%, and 75% concentration) solutions, respectively. The stellate ganglia were taken for histological examination three weeks after the chemical injection. The results showed that the degenerative changes in the cytoplasm and nucleus of ganglionic cells and intercellular tissue were moderate and relatively moderate after the injection of alcohol and glycerol, respectively. Meanwhile, the stellate ganglia revealed mild, relatively moderate, serious and extremely serious degeneration after injection of 10%, 25%, 50%, and 75% phenol, respectively. In conclusion, we recommend a high concentration of phenol, in the least volume, as a chemical agent for clinical injection in the upper thoracic sympathetic ganglion.

Focal adhesion kinase-dependent apoptosis of melanoma induced by tyrosine and phenylalanine deficiency.

We found previously that restriction of tyrosine (Tyr) and phenylalanine (Phe) inhibited growth and metastasis of B16BL6 murine melanoma and arrested these cells in the G0-G1 phase of the cell cycle. Here, we report that deprivation of these two amino acids in vitro induces apoptosis in B16BL6 and in human A375 melanoma cells but not in nontransformed, neonatal murine epidermal cells or human infant foreskin fibroblasts. Four days after deprivation of Tyr and Phe in vitro, 37% of B16BL6 and 51% of A375 melanoma cells were undergoing apoptosis. Apoptosis was not associated with elevation in intracellular calcium or alteration in p53 or c-myc protein expression. Expression and Tyr phosphorylation of focal adhesion kinase (FAK) were inhibited in both melanoma cell lines by deprivation of Tyr and Phe but not by deprivation of glutamine or serum. Tyr phosphorylation of FAK in Tyr- and Phe-deprived melanoma cells was enhanced within 30 min of refeeding with complete DMEM. FAK protein expression recovered within 60 min, and cell viability recovered within 24 h. Genistein, a tyrosine kinase inhibitor that specifically inhibits Tyr phosphorylation of FAK, did not induce apoptosis in A375 melanoma cells at a concentration of 50 microM. Genistein prevented the recovery of cell viability upon refeeding with Tyr and Phe to previously deprived A375 melanoma cells. These data collectively indicate that apoptosis induced by Tyr and Phe deprivation is FAK-dependent.

Inhibition of B16BL6 melanoma invasion by tyrosine and phenylalanine deprivation is associated with decreased secretion of plasminogen activators and increased plasminogen activator inhibitors.

We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) limitation significantly decreased the metastatic phenotype of B16BL6 melanoma cells in vivo and decreased the in vitro invasion of these cells. To more specifically characterize the effects of Tyr and Phe deprivation we examined the three steps involved in invasion: attachment to host cells and components, elaboration of proteases that degrade basement membranes, and migration of invading tumor cells. Here we report that B16BL6 melanoma cell invasion through growth factor reduced (GFR) Matrigel is significantly decreased by Tyr and Phe deprivation. Tyr and Phe deprivation in vitro decreased the attachment of B16BL6 melanoma cells to GFR Matrigel, heparin sulfate proteoglycans (HSPG), neonatal murine epidermal (NME) cells and the extracellular matrix (ECM) from these cells. These cells also exhibited a decrease in chemotactic response to fetal bovine serum (FBS). Deprivation of these two amino acids decreased the secretion of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) while plasminogen activator inhibitor (PAI)-1 and -2 were increased in these cells. These observations suggest that Tyr and Phe deprivation decreases the in vitro chemotactic and invasive ability of B16BL6 melanoma cells by decreasing attachment and secreted PA activity and by increasing secreted PAIs in these cells.

Influence of tyrosine and phenylalanine limitation of cytotoxicity of chimeric TGF-alpha toxins on B16BL6 murine melanoma in vitro.

Previous research in animals supports the use of tyrosine and phenylalanine (Tyr-Phe) restriction as an adjuvant to the treatment of cancer. In this regard, dietary restriction of Tyr-Phe specifically inhibits the growth of B16BL6 melanoma tumors, dramatically suppresses spontaneous hematogenous metastasis, and modulates the sensitivity of these tumor cells to growth factors. Two chimeric toxins, HB-TGF alpha-PE4EKDEL and TGF alpha-PE4EKDEL, were examined for their toxicity against the B16BL6 melanoma cell line, and the ability of Tyr-Phe limitation to modulate the potential of these toxins was examined. Tyr-Phe limitation significantly enhanced the cytotoxic effects of HB-TGF alpha-PE4EKDEL approximately 10-fold toward B16BL6 melanoma, and free heparin diminished the cytotoxicity of HB-TGF alpha-PE4EKDEL. Although TGF alpha-PE4EKDEL is cytotoxic to this cell line, Tyr-Phe limitation did not effect the cytotoxicity of this toxin. Tyr-Phe limitation inhibited the synthesis and secretion of heparin-binding proteins but did not alter the expression of surface heparan sulfate proteoglycans. These data suggest that cell surface heparan sulfate proteoglycan is a target for binding and execution of the cytotoxicity of HB-TGF alpha-PE4EKDEL and that augmentation of cytotoxicity by Tyr-Phe limitation is due to the inhibition of heparin-binding protein production.

Tyrosine and phenylalanine restriction induces G0/G1 cell cycle arrest in murine melanoma in vitro and in vivo.

Tyr-Phe and Met limitation in vitro inhibited cell proliferation and proliferating cell nuclear antigen (PCNA) expression to a greater extent than serum limitation. Tyr-Phe and serum limitation arrested cells in the G0/G1 phase; Met limitation blocked cells in the G0/G1 and S phases. Tyr-Phe limitation progressively decreased cyclin D1 expression to 30% of control within four days and did not affect expression of cyclin D3 or cyclin-dependent kinase (CDK2, CDK4, and CDK5) expression, Met limitation decreased cyclin D3 expression to 25% of control and CDK2 expression to 32% of control by Day 4 and did not affect expression of cyclin D1, CDK4, and CDK5. Serum limitation inhibited cyclin D1 and cyclin D3 expression to 24% of control after four days and did not effect CDK expression. Expression of two CDK inhibitors, p21WAF1/Cip1 and p27Kip1, was not changed by amino acid or serum limitation. Dietary restriction of Tyr-Phe in mice bearing subcutaneous B16BL6 melanoma tumors decreased tumor growth rate compared with mice fed a normal diet. Tumors from Tyr-Phe-restricted mice exhibited decreased PCNA expression, G0/G1 phase cell cycle arrest, and reduced cyclin D1 expression. These data indicate that decreased tumor growth in vivo associated with dietary restriction of Tyr and Phe is cell cycle specific.

Evaluation of laser and radiofrequency induced dorsal root entry zone lesion for pain control in rats.

BACKGROUND: Dorsal root entry zone (DREZ) lesions have been believed to be effective for control of intractable pain. These lesions are usually made using radiofrequency (RF) technique. Theoretically, laser can provide very fast, precise, reproducible and easy control of photothermal effect, possibly achieving better pain control than RF. The objective here was to learn more about the effect of pain control among the RF, carbon dioxide (CO2) laser and potassium titanyl phosphate (KTP) laser in rats. METHODS: The adult rat was anesthetized and the dorsal spinal cord from C5 to T1 was exposed under a microscope. The DREZ lesions were created in each group of eight including sham, RF thermocoagulation, CO2 laser and KTP laser. The latency of pain withdrawal in the fore-paw by a hot-plate test was recorded before the DREZ lesions and three weeks afterward. RESULTS: The data showed that RF, CO2 and KTP laser could significantly reduce pain in rats (p < 0.05, one-way ANOVA and Dunnett's test). Latencies of pain withdrawal in the fore-paw by the hot-plate test before, and three weeks after, DREZ lesions were 13.9 +/- 1.4 sec and 65.2 +/- 4.9 sec in the RF group, 15.9 +/- 1.4 sec and 59.0 +/- 5.9 sec in the CO2 laser group, and 14.1 +/- 0.9 sec and 64.8 +/- 5.7 sec in the KTP group, respectively. CONCLUSIONS: The density of opioid receptor in DREZ lesions cord showed no significant change three weeks after operation in the sham and CO2 laser groups. It was concluded that DREZ lesions caused by RF, CO2 laser and KTP laser can achieve pain control significantly in the rats. The effect of KTP laser was close to RF, followed by CO2 laser.

Expression and subcellular distribution of basic fibroblast growth factor are regulated during migration of endothelial cells.

Migration of endothelial cells is involved in normal and pathological angiogenesis and in re-endothelialization after vascular injury or rupture of atherosclerotic plaques. Several types of endothelial cells are known to synthesize basic fibroblast growth factor (bFGF); in some of these, migration is increased by exogenous bFGF and inhibited by anti-bFGF antibodies. Using immunocytochemical techniques and RNase protection analysis, we studied endothelial cells from bovine coronary arteries and veins as well as from adrenal microvessels. We found that bFGF mRNA and peptide were present in confluent endothelial cells and were upregulated during migration stimulated by removal of some cells from the monolayer. During migration, extracellular matrix stores of bFGF were depleted, and bFGF immunoreactivity began to accumulate in the cytoplasm of endothelial cells between 2 and 6 hours. After migration had begun, but before the initiation of DNA synthesis, bFGF immunoreactivity increased in the nuclei and nucleoli. Exogenous bFGF stimulated endothelial migration, and antibodies to bFGF markedly inhibited migration, suggesting that an intracrine function of nuclear bFGF is not sufficient for cell migration. In all three types of endothelial cells studied, bFGF was identified as an endogenous regulator, but not as the sole regulator, or migration. Moreover, bFGF expression and subcellular localization were found to be regulated during endothelial cell migration.

Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. A clue to the mechanism of transformation from the stable to the unstable clinical state.

BACKGROUND: The mechanisms responsible for the transformation of stable angina to unstable angina, a major cause of morbidity and mortality, are commonly believed to be plaque rupture and thrombosis. We determined whether additional mechanisms are operative by analyzing the histopathology and immuno-histopathology of coronary plaques retrieved by directional atherectomy of patients with unstable angina in whom no intraluminal thrombus was demonstrated by angiography. METHODS AND RESULTS: The histological findings of atherectomy specimens from 34 patients with unstable angina were compared with those of 24 patients with postangioplasty restenosis, whose lesions are known to be composed of smooth muscle cells (SMCs), and 10 patients with stable angina, whose lesions contain relatively few SMCs. We also studied the expression of acidic and basic fibroblast growth factors (aFGF and bFGF), whose role in the vascular response to injury has been established. Specimens from unstable angina resembled those from postangioplasty restenosis in regard to SMC abundance (scale, 0 to 3; 1.4 +/- 0.9 versus 1.7 +/- 0.9; P = NS), and both differed from those of stable angina. Thrombus and/or hemorrhage occurred in only 34% of patients with unstable angina (compared with 8% of restenosis patients and in none of stable angina patients). Active lesions (defined as lesions (defined as lesions containing one or more of the following: thrombus, hemorrhage, abundant and disorganized SMCs in the presence of loose connective tissue, or inflammatory infiltrate) were observed in 56% of the unstable angina patients and in 50% of the restenosis patients but in none of the stable angina patients. The expression of aFGF and bFGF was detected in 80% to 100% of unstable angina (n = 11) and restenosis (n = 10) specimens but in only 1 of 5 stable angina specimens. CONCLUSIONS: Microscopic evidence of thrombosis and plaque rupture occurred in only one third of unstable angina patients, selected because they had no angiographic evidence of intracoronary thrombus. Moreover, their lesions resembled those of restenosis patients in regard to SMC abundance, lesion activity, and the expression of aFGF and bFGF. Our findings therefore suggest that an alternative mechanism to plaque rupture and thrombus formation may be operative in the precipitation of unstable angina; namely, in a subset of patients, SMC proliferation may lead to gradual plaque expansion and thereby to lumenal narrowing and unstable angina. Our data also suggest a role for aFGF and bFGF in this process.

Cytotoxic effects of vascular smooth muscle cells of the chimeric toxin, heparin binding TGF alpha-Pseudomonas exotoxin.

OBJECTIVE: Smooth muscle cell proliferation appears to be very important in restenosis after angioplasty. A chimeric toxin created by genetically fusing the gene encoding TGF alpha (targets the EGF receptor) to the gene encoding Pseudomonas exotoxin (PE) preferentially kills rapidly proliferating smooth muscle cells. Recently, a heparin binding EGF-like growth factor (HB-EGF) has been identified. The HB domain enhances the mitogenic activity for smooth muscle cells. The purpose of this study was to design a new chimeric toxin, having both heparin binding and EGF receptor binding function, and to determine whether it is more cytotoxic to smooth muscle cells. METHODS: By recombinant DNA techniques, a new chimeric toxin, HB-TGF alpha-PE4EKDEL, was synthesised. Cytotoxic assays were performed by assessing the capacity to inhibit protein synthesis of rat vascular smooth muscle cells. RESULTS: The toxin preferentially killed rapidly proliferating smooth muscle cells (p < 0.025). The HB domain increased the cytotoxicity of the molecule when compared to the other chimeric toxins tested against smooth muscle cells. The cytotoxic effect of the new molecule was significantly decreased by exogenously added heparin (p < 0.05). CONCLUSIONS: The presence of a heparin binding domain increases the smooth muscle cell cytotoxicity of the TGF alpha fusion toxin, perhaps because HB-TGF alpha-PE4EKDEL functions as a molecule with two ligands. It will be important to determine whether the greater smooth muscle cell cytotoxicity that exists in vitro will facilitate the specific targeting and killing of rapidly proliferating cells in vivo.