Biomedical subjects

J Kreuzer

Publications and source records attributed to J Kreuzer.

At least 37 records · Page 2Linked to original sources

PubMed2000

Preclinical study on gene therapy of cervical carcinoma using adeno-associated virus vectors.

Approximately 90% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for future tumor therapy. Using recombinant adeno-associated virus type 2 (AAV-2) vectors, two types of therapeutic genes were expressed in cervical carcinoma cells with the aim of suppressing the E6/E7 oncogenes: (a) antisense E6/E7 and ribozyme genes and (b) the monocyte chemoattractant protein-1 (MCP-1) gene encoding MCP-1. Previous studies have shown that the MCP-1 protein is able to indirectly repress E6/E7 gene expression and is consistently absent in tumorigenic HPV-positive cervical carcinoma cell lines. Here, the effect of these therapeutic genes on tumor formation is analyzed in nude mice after ex vivo gene transfer into a HPV16- or HPV18-positive cervical carcinoma cell line (HeLa or SiHa, respectively). Whereas AAV-2 vector-mediated transfer of antisense or even ribozyme genes did not significantly influence tumor formation from implanted SiHa cells, the transfer and expression of human MCP-1 strongly inhibited the development of tumors derived from either HeLa or SiHa cells. Similar results were also obtained after in vivo delivery of these genes into SiHa-derived tumors. This suggests that transfer of therapeutic genes mediating a systemic effect via recombinant AAV-2 vectors offers a promising approach for the development of gene therapies directed against papillomavirus-induced human cancers.

Animals↗

PubMed2000

JNK and c-Jun but not ERK and c-Fos are associated with sustained neointima-formation after balloon injury.

BACKGROUND: Formation of neointima after balloon angioplasty is regulated via inducible transcription factors (ITF), such as c-Jun and c-Fos, depending on mitogen activated protein (MAP) kinases, which have been shown to be activated after balloon injury. The precise localization of activated MAP-kinases and concomitant expression of transcription factors in the vessel wall remains to be elucidated. We have now studied the localization and time-dependent expression of MAP-kinases together with corresponding ITFs in the rat carotid angioplasty model. DESIGN: Animals were sacrificed at 0. 5, 6 and 24 h and 3, 5, 7, 14 and 28 days after injury. Cryocut sections were stained using antibodies directed against c-Jun, phosphorylated c-Jun, c-Fos, c-Jun amino-terminal kinase (JNK), extracellular signal related kinase (ERK), von Willebrand factor, ki67 antigen, and alpha-actin. RESULTS: c-Jun expression was strongly induced in smooth muscle cells (SMC) 30 min after injury and remained upregulated for 24 h, thereafter dropping to basal levels at day 3. Re-expression was observed at day 7 and 14 but not day 28. Expression patterns of JNK and phosphorylated c-Jun were highly congruent to that of c-Jun. In contrast, c-Fos expression was restricted to 30 min and, less pronounced than c-Jun and JNK, was visible after 7 days. Also, its expression was congruent with the presence of ERK. CONCLUSIONS: These findings demonstrate a clear association between MAP-kinases and their transcription factor substrates in vivo with a predominant association of JNK and c-Jun with sustained SMC proliferation.

Animals↗

PubMed2000

The terminal complement complex C5b-9 stimulates interleukin-6 production in human smooth muscle cells through activation of transcription factors NF-kappa B and AP-1.

Activation of the complement system plays an important role in the pathogenesis of atherosclerosis. The proinflammatory cytokine interleukin (IL)-6 is potentially involved in the progression of the disease. We therefore investigated whether the terminal complement complex C5b-9 affects IL-6 production from vascular smooth-muscle cells (VSMC) and set out to determine the underlying signal transduction pathway. Stimulation of human VSMC with C5b-9 resulted in an increase of IL-6 transcript and production of IL-6 protein. Pretreatment with pertussis toxin or pyrrolidine dithiocarbamate inhibited complement-dependent IL-6 mRNA expression and IL-6 release, suggesting the involvement of Gi-proteins and nuclear factor-kB (NF-kB). C5b-9 also induced formation of reactive oxygen species, which, along with IL-6 release, was inhibited by the antioxidant N-acetylcysteine. C5b-9 activated the redox-sensitive transcription factors NF-kB and activator protein-1 (AP-1), which were both involved in the induction of IL-6 by C5b-9, as demonstrated by cis element double-stranded (decoy) oligonucleotides (ODN). The results demonstrate that activation of the complement system induces IL-6 release from human VSMC by a Gi-dependent pathway involving the generation of oxidative stressand the activation of the redox sensitive transcription factors NF-kB and AP-1. Our data support a new mechanism for the proatherogenic effect of the terminal complement complex.

Acetylcysteine↗

PubMed2000

Heparin-mediated selective release of hepatocyte growth factor in humans.

AIMS: The aim of this investigation was to compare the effects of standard (S) with low molecular weight (LMW) heparin on circulating levels of heparin-binding growth factors (HBGF), known to have angiogenic properties in humans. METHODS: In two consecutive trials 18 healthy male volunteers were studied on three separate occasions, following a placebo-controlled crossover design. Subjects were randomised to receive either S-heparin or LMW heparin or placebo. Heparins were administered either by intravenous (i.v.) or subcutaneous (s.c.) injection and saline placebo by i.v. injection. Serum concentrations of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) were measured before and up to 24 h after injection. RESULTS: Administration of i.v. S-or LMW-heparin (50 IU kg(-1) resulted in rapid, highly significant (47 fold for S, 30.9 fold for LMW) increases in HGF serum values, reaching maxima of 10.51+/-1.65 ng ml(-1) (S) and 8.28+/-1.04 ng ml(-1) (LMW), respectively, 10 min after drug application. S.c. injection of S-heparin or LMW heparin resulted in 4.1 and 5.4 fold increases in HGF serum values, respectively. Both agents showed no effects on circulating VEGF or bFGF levels, independent of the route of administration. CONCLUSIONS: Circulating HGF levels were selectively increased in response to pharmacological doses of two, widely used heparin preparations. This may, in part, explain some of the biological effects of heparin separate from its anticoagulant properties. By this mechanism, the systemic administration of heparin may facilitate collateral vessel formation in various clinical settings of tissue ischaemia.

Adult↗

PubMed2000

Efficacy of single lead VDD pacing in patients with impaired and normal left ventricular function.

Atrial synchronous ventricular pacing seems to be the best pacing mode for patients with advanced AV block and impaired LV function. The long-term follow-up of single lead VDD pacing was studied in 33 patients with impaired LV function and compared to 42 patients with normal LV function. All patients received the same VDD lead and VDDR pacemaker. The lead model with 13-cm AV spacing between the atrial and ventricular electrode was implanted in 89% of the patients. Follow-ups were 1, 3, 6, and 12 months after implantation. The percentage of atrial sensing and the P wave amplitude were determined at each follow-up. Minimal P wave amplitude at implantation was 2.0 +/- 1.4 mV in patients with impaired and 1.7 +/- 0.9 mV with normal LV function (not significant). At the 12-month follow-up, 33 patients with normal and 23 patients with depressed LV function remained paced in the VDD mode. The remaining patients died in five (impaired LV function) and seven cases (normal LV function) or their pacemakers were programmed to the VVI/VVIR pacing mode in four (impaired LV function) and three cases (normal LV function). P wave amplitude did not differ in the two groups (e.g., at month 12: impaired: 1.17 +/- 0.42 mV; normal: 1.09 +/- 0.49 mV). The atrial sensitivity was programmed in most patients to sensitive settings with no differences between the two groups (e.g., at month 12: impaired: 0.13 +/- 0.06 mV; normal: 0.13 +/- 0.05 mV). The diagnostic counters indicated nearly permanent atrial sensing (e.g., at month 12: impaired: 99.3 +/- 2.2%; normal: 99.0 +/- 1.0 mV). In conclusions, single lead VDD pacing restored AV synchronous ventricular pacing in patients with normal and with impaired LV function indicating that it could be an alternative to DDD pacemakers, but not to dual-chamber pacing.

Aged↗

PubMed2000

Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species.

The atherogenic effect of the renin-angiotensin system can be explained, in part, by the influence of its effector, angiotensin II (Ang II), on vascular smooth muscle cell (VSMC) growth. There is evidence that reactive oxygen species (ROS) play a role in the atherogenesis and activation of mitogen-activating protein (MAP) kinases, which are involved in proliferation and differentiation. The study was performed to further characterize the role of ROS in Ang II-mediated MAP kinase activation and the regulation of the transcription factor activator protein-1 (AP-1). Rat VSMCs were stimulated with Ang II. The activities of MAP kinases were assessed by Western blot analysis or by immunocomplex kinase assay. AP-1 binding was determined by using an electrophoretic mobility shift assay. Rat VSMCs were treated with Ang II-activated MAP kinases, extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK), p38 MAP kinase (p38 MAPK), and their downstream effector, AP-1. Interestingly, only the activation of ERK1/2, but not JNK or p38 MAPK, was tyrosine kinase, protein kinase C, and MEK1/2 dependent. Ang II also induced the rapid formation of ROS, which could be inhibited by a specific antibody as well as by antisense against the p22phox subunit of the NAD(P)H oxidase. JNK and p38 MAPK, but not ERK, activation was inhibited by an inhibitor of NAD(P)H oxidase. Antisense against p22phox also solely inhibited p38 MAPK but did not affect ERK. The results indicate that in VSMCs, Ang II activates MAP kinases and AP-1 through different pathways; the results further suggest that ROS, generated by p22phox, mediate Ang II-induced JNK and p38 MAPK activation, which may contribute to the pathogenesis of atherosclerosis.

Angiotensin II↗

PubMed2000

Endothelin-1 and smooth muscle cells: induction of jun amino-terminal kinase through an oxygen radical-sensitive mechanism.

Endothelin-1 (ET-1) has been proposed to contribute to atherogenesis and plaque rupture in coronary heart disease through activation of mitogen-activated protein kinases (MAPKs) in smooth muscle cells (SMCs). Reactive oxygen species (ROS) have been shown to be important signal transduction molecules in SMCs. Thus, the present study aimed to assess the role of ROS in ET-1-mediated activation of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2. Rat SMCs were exposed to ET-1 over time at concentrations from 10(-6) to 10(-10) mol/L, and MAPK activity was quantified. Activation of JNK and ERK was observed with a maximum stimulation at 10(-7) mol/L ET-1. JNK and ERK were activated by ET-1 binding to a single receptor (ET-1A) but differed in their downstream mechanisms: only JNK activation was sensitive to the radical scavenger N-acetylcysteine and diphenylene iodonium, an inhibitor of NADPH oxidase, indicating a role for ROS. The downstream MAPK effector and proinflammatory transcription factor, the activator protein-1 complex, was maximally activated 2 hours after the addition of ET-1. It was mainly composed of the JNK substrate c-Jun, and activation was also dependent on ROS formation. We suggest that plaque activation by ET-1 can be mediated through ROS. It can be hypothesized that the clinical benefit of antioxidants in the treatment of atherogenesis may partially depend on neutralization of ET-1-mediated ROS production.

Animals↗

PubMed1999

Effects of HMG-CoA reductase inhibition on PDGF- and angiotensin II- mediated signal transduction: suppression of c-Jun and c-Fos in human smooth muscle cells in vitro.

Hydroxymethylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors were shown to be effective in primary and secondary prevention of coronary heart disease. The beneficial effect of statins is generally attributed to their cholesterol lowering activity. However recent work points to additional cholesterol independent effects of these drugs on cellular signal transduction. In this study it was investigated whether HMG-CoA reductase inhibition could affect induction of the transcription factors c-Jun and c-Fos in smooth muscle cells, which play an important role in atherogenesis. SMC were preincubated for 12 h with or without lovastatin (5 microM) and subsequently stimulated with platelet derived growth factor (PDGF, 10 ng/ml) or angiotensin II (0.1 microM) for 1, 2, 4 and 12 h or with phorbol myristate acetate (100 pM) for 2 h. Stimulation in the absence of the HMG-CoA reductase inhibitor led to a significant induction of c-Jun and c-Fos. Lovastatin inhibited, PDGF-, angiotensin II- and PMA-mediated induction. Concomitant addition of mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate prevented the effects of HMG-CoA reductase inhibition resulting in rescued expression of c-Jun and c-Fos. The suppression of these transcription factors was associated with a complete growth arrest. Viability was not affected by pretreatment with the HMG-CoA reductase inhibitor. The data demonstrate that lovastatin can suppress PDGF- and angiotensin II-mediated induction of c-Jun and c-Fos protein in human SMC. This inhibitory effect may prevent activation of numerous growth factor- and cell cycle- genes. Whether these findings contribute to the effects of statins in atherosclerosis remains to be further investigated.

Angiotensin II↗

PubMed1999

[Primary and secondary prevention of coronary heart disease: what can we afford?].

In secondary prevention, statins are cost-effective and even more cost-effective than other lipd lowering drugs. Statins are especially favorable in patients with previous myocardial infarction compared to those with angina pectoris. For secondary prevention of coronary heart disease, statins should be administered generously. In primary prevention, statins are effective but not cost-efficient. Unfavorable results have to be expected especially in premenopausal women, younger patients, and in those with a low risk factor profile. For population based primary prevention, only dietary measures can be advocated; however, apart from a serious lack of compliance their efficiency has not yet been proven. Preventive measures have their own prize; in general they are considered not to prevent coronary heart disease but retard it. Therefore, such therapeutic interventions can be assumed not to reduce but rather to increase the "direct" medical costs. Furthermore, successful prevention of coronary heart disease leads, due to life-prolongation, to an increase in people out of work and in disabled patients due to age and/or disease. Therefore, an increase especially of the "indirect" costs for unemployment, pension, nursing homes, etc. must be expected.

Coronary Disease↗

PubMed1999

Expression of monocyte chemoattractant protein-1 cDNA in vascular smooth muscle cells: induction of the synthetic phenotype: a possible clue to SMC differentiation in the process of atherogenesis.

In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. Expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for monocytes, has been shown to be among the earliest events in atherogenesis. We investigated the effect of MCP-1 on differentiated and dedifferentiated SMC. Differentiation of SMC was induced using Matrigel as a matrix for cultivation. MCP-1 was expressed in SMC by means of a recombinant adenovirus. Expression of MCP-1 led to dedifferentiation of SMC as demonstrated by induction of cytokeratin 18, a marker for the synthetic phenotype. Concurrently, migration was only detectable in MCP-1 expressing cells, whereas SMC infected with a control virus, coding for the nuclear-targeted lacZ gene showed no migration. The expression of intercellular adhesion molecule-1 (ICAM-1) could be demonstrated in synthetic SMC and was induced after infection of differentiated cells with recombinant adenovirus, coding for MCP-1 (AdMCP-1). Expression of ICAM-1 was associated with a tenfold higher monocyte binding compared to lacZ infected cells. Our data suggest that MCP-1 plays an important role for SMC in the functional switch from the contractile to the synthetic phenotype in the course of atherogenesis.

Adenoviridae↗

PubMed1999

3-deazaadenosine prevents adhesion molecule expression and atherosclerotic lesion formation in the aortas of C57BL/6J mice.

Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) play an important role during the development of atherosclerosis. 3-Deazaadenosine (c(3)Ado), an adenosine analogue, inhibits endothelial-leukocyte adhesion and ICAM-1-expression in vitro. We hypothesized that c(3)Ado is able to prevent the expression of adhesion molecules and atherosclerotic lesion formation in female C57BL/6J mice. The animals were placed on an atherogenic diet with or without c(3)Ado for 9 weeks. Frozen cross sections of the proximal ascending aorta just beyond the aortic sinus were stained with oil red O, hematoxylin, and elastic van Gieson's stains and were analyzed by computer-aided planimetry for fatty plaque formation and neointimal proliferation. Monoclonal antibodies against CD11b (macrophages), VCAM-1, and ICAM-1 were used for immunohistochemistry. Mice on the atherogenic diet demonstrated multiple (5.4+/-1.6 per animal) lesions covering 3.4+/-2.8% of the endothelium and a marked neointima when compared with control mice (4501+/-775 versus 160+/-38 microm(2), P<0.001). Mice on the cholesterol-rich diet without c(3)Ado showed strong endothelial coexpression of ICAM-1 and VCAM-1. Moreover, there was a 10-fold increase in monocyte accumulation on the endothelial surface (33. 3+/-4.9 versus 3.8+/-1.2, P<0.004). In contrast, in mice treated with c(3)Ado, expression of ICAM-1 and VCAM-1 as well as monocyte adhesion and infiltration were almost completely inhibited. Furthermore, these mice did not show any fatty streak formation or neointima formation (125+/-32 microm(2)). Our results demonstrate that c(3)Ado can inhibit diet-induced fatty streak formation and the expression of endothelial ICAM-1 and VCAM-1 in C57BL/6J mice. This may provide a novel pharmacological approach in the prevention and treatment of atherosclerosis.

Animals↗

PubMed1999

The lipoprotein lipase HindIII polymorphism: association with total cholesterol and LDL-cholesterol, but not with HDL and triglycerides in 342 females.

BACKGROUND: Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of core triglycerides in chylomicrons and VLDL. METHODS: We investigated the association between the HindIII polymorphism of the LPL gene and fasting glucose, lipid, and lipoprotein concentrations in 683 Caucasians. We first stabilized the study subjects, using an 8-day diet and exercise intervention program before obtaining blood samples. The use of this standardization period reduced the variance of all glucose and lipid concentrations. RESULTS: In our study, the HindIII allele frequencies for females and males were 0.29 and 0.34 for H- and 0.71 and 0.66 for H+, respectively. We found in females, but not in males, a significant association between the HindIII genotype and total cholesterol (P = 0.007) and LDL-cholesterol (P = 0.018), with females homozygous for the rare H- allele having the lowest, heterozygotes (H-/+) having intermediate, and women homozygous for the common H+ allele having the highest of each of these lipid traits. With regard to triglycerides, HDL-cholesterol, and glucose, no significant effect of the HindIII genotype was noted in either gender. CONCLUSIONS: These results suggest that in a gender-specific manner, the rare LPL HindIII H- allele has a cholesterol-lowering and, therefore, potentially cardioprotective effect compared with the common H+ allele.

Alleles↗

PubMed1998

[The intraoperative measured stimulation impedance of pacemaker electrodes is not a predictor for their longevity].

UNLABELLED: As pacing impedance is inversely related to pacing current, the increase of pacing impedance additionally decreases pacing current. Whether the impedance measurement at implantation predicts the outcome during follow-up, was studied in 87 patients who received the VDD-single lead UniPass 425 connected to the pacemaker Unity (Sulzer Intermedics). The impedance changes between implantation and 6 months follow-up were assessed for each patient. Similar impedance values were defined, if the two measurements were within a range < or = -100 to +100 omega. Six-months impedance was lower or higher compared to implantation, if the difference exceeded > -100 or > +100 omega. At implantation, impedance was 535 +/- 98 omega (range: 333-811 omega) and significantly increased to 604 +/- 160 omega (range: 361-1150 omega) after 6 months. Mean difference between the two measurements was 69 +/- 162 omega (range: -336 bis + 560 omega). Similar impedance had 43 (implantation: 527 +/- 75 omega, 6 months: 531 +/- 87 omega), lower values 11 (implantation: 660 +/- 83 omega, 6 months: 494 +/- 73 omega) and higher values 33 patients (implantation: 503 +/- 99 omega, 6 months: 735 +/- 168 omega). Compared to the patients with similar impedance patients with lower impedance had a significantly higher impedance values at implantation. CONCLUSIONS: Pacing impedance increased significantly within 6 months after implantation. Pacing impedance changed > 100 omega in 51% of the patients. The long-term follow-up of pacing impedance can be predicted generally, but not for the individual patient.

Aged↗

PubMed1998

Acute effects of haemodialysis on cutaneous microcirculation in patients with peripheral arterial occlusive disease.

BACKGROUND: Peripheral arterial occlusive disease (PAOD) is an increasing problem in patients on maintenance haemodialysis. Alterations in microvascular perfusion accompany and complicate arteriosclerosis of large vessels and might contribute to the disease process. The aim of the study was to investigate the acute effects of haemodialysis on the cutaneous microcirculation in 26 patients with and without intermittent claudication. METHODS: Cutaneous perfusion was assessed by measuring transcutaneous oxygen pressure (tcPO2) and skin temperature at the dorsum of the foot. After standardized cooling to 15 degrees C of a 2cm2 skin area, the time to reach baseline skin temperature was evaluated as an indirect parameter of reactive hyperaemia. RESULTS: During haemodialysis, tcPO2 dropped significantly in both groups. The decrease in tcPO2 was more pronounced in patients with PAOD (20% vs 15% n.s.). The reactive hyperaemia response was reduced significantly in patients with intermittent claudication indicated by a prolonged time to reach baseline skin temperature after cooling. Values of tcPO2 and reactive hyperaemia did not reach baseline values at the end of haemodialysis in either group. CONCLUSIONS: Nutritive skin perfusion is impaired during haemodialysis. These changes are more pronounced in patients with PAOD and persist after dialysis. These findings are relevant for the treatment of patients with vascular disease on maintenance haemodialysis.

Adult↗

PubMed1997

[The effect of a lower stimulation frequency on the AV synchronicity of VDD pacemakers].

BACKGROUND AND OBJECTIVE: Implantation of a VDD pacemaker (ventricular pacing; dual sensing [atrial and ventricular]; dual response [triggered + inhibited]) together with a single VDD electrode catheter restores synchronous AV ventricular stimulation in patients with higher-grade AV block and intact sinus function. If higher-frequency stimulation occurs it may be a sign of pacemaker malfunction or of inadequate pacemaker programming. This study was undertaken to determine, at first follow-up examination, in how many patients with a VDD pacemaker VVI stimulation occurred more than 5% of the time; how such patients differed from those with 5% or fewer VVI stimulations; and whether a changed program reduced the proportion of VVI stimulations. PATIENTS AND METHODS: 67 consecutive patients were tested 1 to 3 months after implantation of the Unity VDD pacemaker (Sulzer Intermedics). The frequency of VVI stimulations was determined via a diagnostic pacemaker memory store. After intermediate analysis, programming was optimized and the patients then re-tested 12 months after the initial implantation. RESULTS: At the first follow-up examination 54 patients had VVI stimulations of < or = 5% (0.5 +/- 0.9%) and 13 had > 5% of the time (19.8 +/- 10.7%). The two groups differed significantly from one another in their lower intervention frequency (< or = 5% VVI stimulations: 47 +/- 6/min; > 5% VVI stimulations: 58 +/- 5/min). In particular, the pacemakers in patients with > 5% VVI stimulations had been significantly more often programmed to values of > 50/min. As a result, the pacemakers of these patients were reprogrammed to a lower frequency. A year after implantation there was no longer any difference in the lower intervention frequency, 44 +/- 4/min, between patients with initially > 5% VVI stimulations and those with initially < or = 5% stimulations. At the same time, the proportion of VVI stimulations fell to 4 +/- 6%, with 67% of patients having AV synchronicity of > 95%. INTERPRETATION: At first follow-up, patients with > 5% VVI stimulations differed from those with < or = 5% stimulations with regard to an increased lower intervention frequency. In most of these patients the proportion of AV stimulations was increased to > 95% by reducing the lower intervention frequency to < or = 50/min.

Aged↗

PubMed1997

Protein kinase C activation after cellular adhesion on fibronectin: partial suppression after inhibition of protein isoprenylation.

The cellular events following interaction between matrix proteins and cells are important requisites for physiological mechanisms as well as the progress of a number of diseases. Cellular adhesion to fibronectin, an important component of the extracellular matrix has been demonstrated to be associated with translocation of protein kinase C (PKC) by an integrin-dependent pathway. For this process G-proteins may play an important role as coupling proteins. Membrane association and activity of G-proteins has been shown to be regulated by isoprenylation. We therefore studied whether fibronectin mediated adhesion resulted in PKC translocation and if isoprenylation of cellular proteins may play a role for this integrin-dependent pathway of PKC activation. Chinese hamster ovary (CHO) cells were pretreated with either the Hydroxy-methylglutaryl(HMG)-CoA reductase inhibitor lovastatin or prenylation inhibitor limonene. For the stimulation by extracellular matrices, CHO cells were plated on tissue culture dishes coated with fibronectin or bovine serum albumin and PKC activity was determined. To investigate direct effects of inhibition of isoprenylation on cytoskeletal organization, phalloidin-stained stress fibers were characterized after adhesion on different matrices. CHO cells seeded on fibronectin displayed over twice the PKC translocation to the particulate fraction in comparison to that measured in cells on albumin. Pretreatment of CHO cells with lovastatin or limonene resulted in partial suppression of PKC activation after cell-seeding on the specific matrix fibronectin. Changed PKC distribution was not due to a disorganization of the actin skeleton. These data show that inhibition of isoprenylation of cellular proteins, possibly small Guanosine triphosphate(GTP)-binding proteins, alters only the integrin-mediated PKC distribution but does not greatly influence constitutive PKC distribution.

Animals↗

PubMed1997

Amino terminus of apolipoprotein B suffices to produce recognition of malondialdehyde-modified low density lipoprotein by the scavenger receptor of human monocyte-macrophages.

Malondialdehyde, a product of lipid peroxidation, produces threshold conversion of low density lipoprotein (LDL) to a form recognized by type I and type II scavenger receptors of monocyte-macrophages. To investigate whether localized domains of human apoB-100 protein provide recognition determinants, we tested the ability of several different apoB-bearing particles to interact with the scavenger receptor of human monocyte-macrophages. Genetically engineered, carboxyl-terminally truncated apoB proteins assembled into lipoprotein form were labeled by fluorescent dye. Fluorescence microscopy and quantitative fluorescent spectrophotometry showed that purified particles containing as little as 23% of the apoB amino-terminus were internalized by the scavenger receptor after, but not before, malondialdehyde modification. There was no recognition of the particles by the LDL receptor. Similar results were obtained with human plasma LDL homozygous for carboxyl-terminally truncated apoB-45.2. Liposome-incorporated fusion protein containing apoB residues 547-735 displayed specific uptake by the scavenger receptor without modification by malondialdehyde. In contrast, fusion proteins containing apoB residues 3,029-3,133 or a short amino terminal segment failed to interact. Thus, primary sequence presented by residues 1-1,084 sufficed to produce recognition of modified LDL by the scavenger receptor. These receptor-combining domains were sequestered when secreted in lipoprotein form and were expressed upon malondialdehyde modification. When packaged exogenously in liposome form, fusion protein containing apoB residues 547-735, containing approximately 4% of the primary sequence, mediated scavenger receptor-dependent uptake and hydrolysis. Our findings provide an additional function or the amino-terminal region of apoB and demonstrate that primary sequence presented by the first 2% of apoB-100 protein suffices to produce recognition on malondialdehyde-modified LDL by the scavenger receptor of human monocyte-macrophages.

Amino Acid Sequence↗

PubMed1996

Predictive value of lipid profile for salutary coronary angiographic changes in patients on a low-fat diet and physical exercise program.

In this study, 113 patients with modestly elevated levels of low-density lipoprotein cholesterol (<210 mg/dl) and coronary artery disease were randomized to an intervention group (n=56) or a control group (n=57). The intervention program consisted of daily exercise and a low-fat diet according to the American Heart Association's recommendation phase III; patients in the control group received "usual care" rendered by their private physician. After 1 year, complete data were available for all 92 patients (intervention: n=40; control: n=52) who underwent repeat coronary angiography. During the study course, patients in the intervention group showed an increase in apolipoprotein A-I(123 +/- 18 vs 129 +/- 20 mg/dl; p < 0.02) and apolipoprotein A-I/B (1.3 +/- 0.4 vs 1.5 +/- 0.4; p <0.01) and a decrease in apolipoprotein B (99 +/- 20 vs 89 +/- 18 mg/dl; p < 0.01), while apolipoprotein A-II remained unchanged (38 +/- 6 vs 38 +/- 6 mg/dl; p=NS). In the control group, there were no significant changes (apolipoprotein A-I, 124 +/- 17 vs 128 +/- 13 mg/dl; apolipoprotein A-II, 38 +/- 6 vs 39 +/- 6 mg/dl; apolipoprotein B, 100 +/- 21 vs 99 +/- 16 mg/dl; apolipoprotein A-I/B, 1.3 +/- 0.3 vs 1.4 +/- 0.5; all p=NS). As previously reported, there was a significant retardation of progression in patients in the intervention group (progression 23%, no change 45%, regression 32%) compared with the control group (progression 48%, no change 35%, regression 17%) (p < 0.05). Although retardation of progression was significantly associated with an increase in apolipoprotein A-I/B and a decrease in apolipoprotein B (p < 0.05), these gave way in multivariate analysis to changes in total cholesterol/high-density lipoprotein cholesterol, absolute levels of low-density lipoprotein cholesterol, and, in a subgroup of patients, to leisure-time physical activity (all p < 0.05). These data demonstrate that an intervention based on a low-fat diet and intensive physical exercise is capable of improving apolipoprotein levels, associated with retardation of progression of coronary artery disease. However, total cholesterol/high-density lipoprotein cholesterol and low-density lipoprotein cholesterol appear superior to apolipoproteins as metabolic markers for effective treatment in patients with coronary artery disease.

Adult↗