The effect of radiation dose on the tensile and impact toughness of highly cross-linked and remelted ultrahigh-molecular weight polyethylenes.

Several highly cross-linked and remelted ultrahigh-molecular weight polyethylenes (UHMWPE) were introduced in 1998 as bearing materials for orthopaedic implants to achieve superior wear performance. However, gamma radiation and the subsequent postirradiation thermal treatment are associated with decreased mechanical properties such as ductility, toughness, and fatigue strength compared to noncross-linked materials. The purpose of this study was (a) to characterize the toughness (tensile and impact) of highly cross-linked and remelted UHMWPE of varying doses (0-255 kGy) and (b) determine whether a correlation exists between both toughness measures. As radiation dose increased, tensile toughness and impact toughness were shown to decrease in a nonlinear fashion; largely a result of the decrease in ductility, which was also observed. Impact toughness and tensile toughness were also found to be strongly correlated to one another (R(2) = 0.97).

Evaluating the suitability of highly cross-linked and remelted materials for use in posterior stabilized knees.

Posterior stabilized (PS) knee designs are a popular choice for cruciate sacrificing knee arthroplasty procedures. The introduction of PS inserts fabricated from highly cross-linked and remelted Ultra High Molecular Weight Polyethylene (UHMWPE) has recently generated concern as these materials have been shown to possess reduced mechanical properties. This study investigated whether highly cross-linked and remelted UHMWPE material (referred to as XRP) can be expected to perform similarly to historical gamma-air polyethylene, which has suffered few reported incidences of tibial post failure. Never-implanted gamma-air PS tibial inserts shelf-aged 14 years were examined and compared to XRP materials. Evaluation of oxidation levels, impact toughness, and fatigue strength demonstrated never-implanted gamma-air PS tibial inserts to possess nonuniform mechanical properties. Despite severe oxidation along the exterior of gamma-air tibial posts, comparatively low oxidation levels at the center of the tibial posts corresponded to sufficiently high mechanical properties. XRP material (75 kGy) showed superior impact toughness over shelf aged gamma-air material; however, tibial post fatigue testing demonstrated XRP material (100 kGy) to be less resistant to fatigue failure than historical gamma-air material. Results from this study indicate that XRP materials (100 kGy) may demonstrate an inferior resistance to tibial post failure than historical polyethylene.

Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases.

The invasive properties of cancer cells depend on their intrinsic motile potential and on their ability to breach the endothelial barrier. In the present work, we investigated the mechanisms by which adhesion of colon cancer cells to E-selectin expressed by endothelial cells regulates the barrier function of these cells and modulates transmigration of cancer cells. We found that the stimulation of E-selectin by activating antibodies or the adhesion of HT-29 cells results in an increase in the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases. In turn, the activation of p38 and ERK enhances transendothelial permeability and migration of HT-29 cells. We also obtained evidence suggesting that p38-mediated increase in transendothelial migration of cancer cells depends on a myosin light chain phosphorylation-mediated formation of stress fibres. On the other hand, the activation of ERK by E-selectin modulates the opening of interendothelial spaces by initiating the activation of Src kinase activities and the dissociation of the VE-cadherin/beta-catenin complex. We conclude that activation of E-selectin by adhering cancer cells is an important process that regulates the extravasation of colon cancer cells by initiating p38- and ERK-dependent mechanisms that both contribute to regulate the integrity of the endothelial layer.

Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activated protein kinase-2 (SAPK2/p38) in the tumor cells.

Adhesion and migration of tumor cells on and through the vascular endothelium are critical steps of the metastatic invasion. We investigated the roles of E-selectin and of stress-activated protein kinase-2 (SAPK2/p38) in modulating endothelial adhesion and transendothelial migration of HT-29 colon carcinoma cells. Tumor necrosis factor alpha (TNF alpha) strongly increased the expression of E-selectin in human umbilical vein endothelial cells (HUVEC). This effect was independent of the activation of SAPK2/p38 induced by TNF alpha. Adhesion of HT-29 cells on a monolayer of HUVEC pretreated with TNF alpha was dependent on E-selectin expression but was independent of SAPK2/p38 activity of both HUVEC and tumor cells. The adhesion of HT-29 cells to E-selectin-expressing HUVEC led to the activation of SAPK2/p38 in the tumor cells as reflected by the increased phosphorylation of the actin-polymerizing factor HSP27 by mitogen-activated protein kinase 2/3, a direct target of SAPK2/p38. Moreover, a recombinant E-selectin/Fc chimera quickly increased the activation of SAPK2/p38 in HT-29 cells. Blocking the increased activity of SAPK2/p38 of HT-29 cells by SB203580 or by expressing a dominant negative form of SAPK2/p38 inhibited their transendothelial migration. Similarly, HeLa cells stably expressing a kinase-inactive mutant of SAPK2/p38 showed a decreased capacity to cross a layer of HUVEC. Overall, our results suggest that the regulation of transendothelial migration of tumor cells involves two essential steps as follows: adhesion to the endothelium through adhesion molecules, such as E-selectin, and increased motogenic potential through adhesion-mediated activation of the SAPK2/p38 pathway.

Involvement of p38 in apoptosis-associated membrane blebbing and nuclear condensation.

The stress-activated protein kinase p38 is often induced by cytotoxic agents, but its contribution to cell death is ill defined. In Rat-1 cells, we found a strong correlation between activation of p38 and induction of c-Myc-dependent apoptosis. In cells with deregulated c-Myc expression but not in control cells, cis-diamminedichloroplatinum induced p38 activity and typical features of apoptosis, including internucleosomal DNA degradation, induction of caspase activities, and both nuclear (nuclear condensation and fragmentation) and extranuclear (cell blebbing) morphological alterations. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not block p38 activation and the p38 inhibitor SB203580 had no detectable effect on the activation of caspases or the in vivo cleavage of several caspase substrates, suggesting that p38 and caspase activation can contribute distinct features of apoptosis. Accordingly, we found that cell blebbing was independent of caspase activity and, rather, depended on p38-sensitive changes in microfilament dynamics likely mediated by heat shock protein 27 phosphorylation. Furthermore, p38 activity contributed to both caspase-dependent and caspase-independent nuclear condensation and fragmentation, suggesting a role in an early event triggering both mechanisms of apoptosis or sensitizing the cells to the action of both types of apoptosis executioners. Inhibiting p38 also resulted in a significant enhancement in cell survival estimated by colony formation. This capacity to modulate the sensitivity to apoptosis in cells with deregulated c-Myc expression suggests an important role for p38 in tumor cell killing by chemotherapeutic agents.

Markers of neck failure in oral cavity and oropharyngeal carcinomas treated with radiotherapy.

BACKGROUND: Neck management after radiotherapy remains controversial. It is not clear which patients may benefit from postradiotherapy neck dissection. Biologic markers may be useful in this setting. METHOD: This study includes 81 patients with oral cavity and oropharyngeal carcinomas. The primary tumor had been treated with radical radiotherapy. Immunohistochemical staining to p53, ki-67, NEU, HSP-27, and GST has been performed. RESULTS: There were 50 T1-2 and 31 T3-4 patients, as well as 36 NO and 45 N1-3. A total of 25 nodal failures was observed. With expressed HSP2, 23% of patients had neck failure compared with 51% when HSP-27 was absent (p = .02). With NEU overexpression, nodal control decreased from 72% to 34% (p = .008). In a Cox model, NEU (p = .01) and HSP-27 (p = .05) were associated with neck failure. CONCLUSIONS: HSP-27 and NEU expression may play a role in predicting nodal failure. This should be confirmed in a larger, prospective study.

p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle.

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT(1) receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H(2)O(2). Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.

A computer-assisted assessment of lifetime physical activity: reliability and validity of the QUANTAP software.

BACKGROUND: This study investigated the reliability and the validity of the QUANTAP (QUANTification de l'Activité Physique) interview-administered survey, a new computer-assisted tool designed to determine physical activity over a lifetime. METHODS: The tool was used to assess lifetime exercise habits in four dimensions (sport at school, leisure sport, occupation, daily activities) in 419 men and women aged 13-90 years. Physical activity indicators (time spent and energy expenditure) were calculated for 20-year periods. The inter-observer and intra-observer reliability of the tool was studied in two subgroups of 30 subjects. RESULTS: Intraclass correlation coefficients for intra-observer and inter-observer reliability varied from 0.56 to 0.96 and from 0.42 to 0.99 respectively according to the dimensions and indicators considered. Energy expenditure was not statistically significantly different from recommended nutritional intake in either males or females. Percent body fat at the time of the survey correlated with leisure sport (particularly in recent periods of practice): age-adjusted correlation coefficients varied from - 0.23 to - 0.45 among males, and from - 0.19 to - 0.31 among females. CONCLUSION: The results indicate that QUANTAP is reliable and valid to assess lifetime physical activity. It therefore provides a tool with which the long-term effects of physical activity on current health may be studied.

Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase.

In endothelial cells, vascular endothelial growth factor (VEGF) induces an accumulation of stress fibers associated with new actin polymerization and rapid formation of focal adhesions at the ventral surface of the cells. This cytoskeletal reorganization results in an intense motogenic activity. Using porcine endothelial cells expressing one or the other type of the VEGF receptors, VEGFR1 or VEGFR2, or human umbilical vein endothelial cells pretreated with a VEGFR2 neutralizing antibody, we show that VEGFR2 is responsible for VEGF-induced activation of the stress-activated protein kinase-2/p38 (SAPK2/p38), phosphorylation of focal adhesion kinase (FAK), and enhanced migratory activity. Activation of SAPK2/p38 triggered actin polymerization whereas FAK, which was phosphorylated independently of SAPK2/p38, initiated assembly of focal adhesions. Both processes contributed to the formation of stress fibers. Geldanamycin, an inhibitor of HSP90 blocked tyrosine phosphorylation of FAK, assembly of focal adhesions, actin reorganization, and cell migration, all of which were reversed by overexpressing HSP90. We conclude that VEGFR2 mediates the physiological effect of VEGF on cell migration and that two independent pathways downstream of VEGFR2 regulate actin-based motility. One pathway involves SAPK2/p38 and leads to enhanced actin polymerization activity. The other involves HSP90 as a permissive signal transduction factor implicated in FAK phosphorylation and assembly of focal adhesions.

Overexpression of the 27 KDa heat shock protein is associated with thermoresistance and chemoresistance but not with radioresistance.

PURPOSE: The heat shock protein (hsp27) correlates with thermotolerance and chemoresistance. Our main objective was to assess the response to radiotherapy both in vitro and in vivo in correlation with various concentrations of hsp27. The second objective was to evaluate the relation between hsp27 and glutathione-s-transferase pi (GST pi). METHODS AND MATERIALS: For the in vitro study, thermoresistant cell lines, expressing various amounts of hsp27, were used to assess the role of this protein in radioresistance. To verify the efficiency of hsp27 in these cells lines to confer resistance to cytotoxic agents, these cells were also treated with heat shock and cisplatin. Furthermore, the role of hsp27 expression was studied in vivo by immunochemistry in 98 patients with head and neck squamous cell carcinoma treated by radiotherapy. hsp27 was correlated with local control of the tumor and with clinical and biologic factors potentially able to affect the local control, including p53, ki-67, ploidy, and GST. RESULTS: In vitro, high constitutive levels of expression of hsp27 did not significantly influence the survival curves of transfected cells exposed to radiation as compared to control cells although hsp27 overexpression was confirmed to increased the cellular resistance to heat and to cisplatinum. In vivo, we showed that overexpression of various amounts of hsp27 did not correlate with local control of the tumor. In vivo, hsp27 was only significantly associated with GST pi. Expression of GST pi was associated with poor local (p = 0.01) control and survival (p = 0.08) in a Cox model. CONCLUSIONS: It is concluded that the mechanisms responsible for hsp27-mediated heat and drug resistance are not involved in radioprotection.

Integrating the VEGF signals leading to actin-based motility in vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor that stimulates endothelial cell proliferation and migration, two key events of the angiogenic process. The intracellular signals leading to these events have recently been investigated and a better understanding on how VEGF induces its angiogenic functions is emerging. Herein, we summarize recent findings on how VEGF stimulates endothelial cell migration and contributes to angiogenesis. In particular, the role of the VEGF receptors involved in initiating the coordinated signals that leads to actin-based motility is discussed.

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent.

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.

A short lived protein involved in the heat shock sensing mechanism responsible for stress-activated protein kinase 2 (SAPK2/p38) activation.

The stress-activated protein kinase 2 (SAPK2/p38) is activated by various environmental stresses and also by a vast array of agonists including growth factors and cytokines. This implies the existence of multiple proximal signaling pathways converging to the SAPK2/p38 activation cascade. Here, we show that there is a sensing mechanism highly specific to heat shock for activation of SAPK2/p38. After mild heat shock, cells became refractory to reinduction of the SAPK2/p38 pathway by a second heat shock. This was not the result of a toxic effect because the cells remained fully responsive to reinduction by other stresses, cytokines, or growth factors. Neither the activity of SAPK2/p38 itself nor the accumulation of the heat shock proteins was essential in the desensitization process. The cells were not desensitized to heat shock by other treatments that activated SAPK2/p38. Moreover, inhibiting SAPK2/p38 activity during heat shock did not block desensitization. Also, overexpression of HSP70, HSP27, or HSP90 by gene transfection did not cause desensitization, and inhibiting their synthesis after heat shock did not prevent desensitization. Desensitization rather appeared to be linked closely to the turnover of a putative upstream activator of SAPK2/p38. Cycloheximide induced a progressive and eventually complete desensitization. The effect was specific to heat shock and minimally affected activation by other stress inducers. Inhibiting protein degradation with MG132 caused the constitutive activation of SAPK2/p38, which was blocked by a pretreatment with either cycloheximide or heat shock. The results thus indicate that there is a sensing pathway highly specific to heat shock upstream of SAPK2/p38 activation. The pathway appears to involve a short lived protein that is the target of rapid successive up- and down-regulation by heat shock.

[Central neurocytoma. A report of 4 cases]. / Le neurocytome central. A propos de 4 observations.

We report 4 cases of central neurocytoma removed by a transfrontal approach with no recurrence after a mean follow up of 3 years. This uncommon lesion of the supratentorial ventricles (150 cases reported) occurs in young adults, and often induces intracranial hypertension secondary to an obstructive hydrocephalus. The CT scan, MRI and histopathological features are related. This neuronal origin tumor is difficult to distinguish from other intraventricular processes as oligodendroglioma or ependymoma. However, the immunopositivity for the neuronal markers as synaptophysin, calcineurin and microtubul associated protein 2, and the negativity for the glial fibrillary acidic protein, allow the diagnosis of this neuropathological entity. The prognosis is favorable though some cases of recurrence (14 cases) and cerebrospinal dissemination (2 cases) has been reported in the literature.

Prediction of body composition of live and post-mortem red foxes.

A series of measurements (lengths, circumferences, skinfolds, masses and resistance) was taken on 29 red foxes (Vulpes vulpes) of both sexes before and after their death during the autumns of 1994 and 1995. Body composition of each carcass was determined by chemical analysis of homogenized samples of viscera, carcass and skin. Eight multiple regression models were then developed to predict body water, fat, protein, and mineral mass using body measurements as independent variables taken on live or dead animals. All final models were highly significant (P < 0.0001) and included three or four explanatory variables. Adjusted coefficients of determination varied between 0.95 for water mass and 0.81 for mineral mass. The models cover a wide range of conditions as percent body fat in the 29 samples varied between 1.1 and 28.4%. Our models can serve for management or research purposes with live or dead red foxes as they are quick, inexpensive and nondestructive.

Regulation of actin dynamics by stress-activated protein kinase 2 (SAPK2)-dependent phosphorylation of heat-shock protein of 27 kDa (Hsp27).

Activation of the mitogen-activated protein kinase (MAP kinase) SAPK2 (stress-activated protein kinase 2) leads to the phosphorylation of several transcription factors and cytoplasmic proteins, and thereby presumably orchestrates important specific cellular responses to numerous cytokines, stressing agents and agonists of tyrosine kinase or serpentine receptors. The heat-shock protein of 27 kDa (Hsp27), a downstream target of the SAPK2-activated MAP-kinase-activated protein kinase-2/3, has a documented function as an actin polymerization modulator. Accordingly, recent evidence implicates the SAPK2 pathway in the modulation of microfilament dynamics in response to stress and agonist stimulation. In vascular endothelial cells, where the basal level of expression of Hsp27 is high, SAPK2 mediates oxidative stress- and vascular endothelial growth factor (VEGF)-induced actin reorganization and VEGF-induced cell migration, suggesting a key role for this MAP kinase pathway in inflammation and angiogenic processes.

Embryonic death of Mek1-deficient mice reveals a role for this kinase in angiogenesis in the labyrinthine region of the placenta.

Mek is a dual-specificity kinase that activates the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. The Erk MAP kinase cascade is involved in cell-fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single mek gene is present in Caenorhabditis elegans, Drosophila and Xenopus, two mek homologs, Mek1 and Mek2, are present in the mammalian cascade. In the present study, we describe a mutant mouse line in which the mek1 gene has been disrupted by insertional mutagenesis. The null mutation was recessive lethal, as the homozygous mutant embryos died at 10.5 days of gestation. Histopathological analyses revealed a reduction in vascularization of the placenta that was due to a marked decrease of vascular endothelial cells in the labyrinthine region. The failure to establish a functional placenta probably explains the death of the mek1-/- embryos. Cell-migration assays indicated that mek1-/- fibroblasts could not be induced to migrate by fibronectin, although the levels of Mek2 protein and Erk activation were normal. Re-expression of Mek1 in the mutant mouse embryonic fibroblasts (MEFs) restored their ability to migrate. Our findings provide genetic evidence that establishes the unique role played by Mek1 in signal transduction. They also suggest that mek1 function is required for normal response to angiogenic signals that might promote vascularization of the labyrinthine region of the placenta.

SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis.

In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization-generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.

Role of the mitogen-activated protein kinases in the expression of the kinin B1 receptors induced by tissue injury.

Several cytokines and LPS regulate the population of the B1 receptors (B1Rs) for kinins; these are responsive to des-Arg9-bradykinin (BK) and Lys-des-Arg9-BK. B1R activation contributes to inflammatory vascular changes and pain. Aortic rings isolated from normal rabbits and incubated in vitro in Krebs physiological medium were used as a model of tissue injury. From a null level of response, these rings exhibit a time- and protein synthesis-dependent increase in the maximal contractile response to des-Arg9-BK. Exposure to exogenous IL-1beta or epidermal growth factor (EGF) considerably increases the process of sensitization to the kinins. Freshly isolated control aortic rings showed high mitogen-activated protein (MAP) kinase activities (persistent activation of p38, but less prolonged for extracellular signal-regulated kinase and c-Jun-N-terminal kinase/stress-activated protein kinase pathways) relatively to the basal activities found in various types of cultured cells. IL-1beta or EGF further increased the activities of the extracellular signal-regulated kinase and c-Jun-N-terminal kinase/stress-activated protein kinase MAP kinases. The inhibitor of the p38 MAP kinase, SB 203580 (10 microM), massively (approximately 75%) and selectively inhibited the spontaneous sensitization to des-Arg9-BK over 6 h. SB 203580 also significantly reduced the development of the response to des-Arg9-BK as stimulated by IL-1 or EGF. Both spontaneous and IL-1beta-stimulated up-regulation of responsiveness to des-Arg9-BK were significantly inhibited by the MAP kinase extracellular signal-regulated kinase kinase 1 inhibitor PD 98059 (approximately 40%). The protein kinase inhibitors failed to inhibit protein synthesis and to acutely inhibit the contractile effect of des-Arg9-BK, suggesting that they do not influence B1 receptor transduction mechanisms. In cultured aortic smooth muscle cells stimulated with EGF, MAP kinase activation preceded B1R mRNA induction. Protein kinase inhibitors reveal the role of cell injury-controlled MAP kinase pathways, and singularly of the p38 pathway, in the induction of B1R.

p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent chemotactic agent for endothelial cells. Yet the signalling pathways that modulate the motogenic effects of VEGF in vascular endothelial cells are still ill defined. In the present study, we found in primary cultures of human umbilical vein endothelial cells (HUVEC) that VEGF increased cell migration and induced a marked reorganization of the microfilament network that was characterized by the formation of stress fibers and the recruitment of vinculin to focal adhesions. VEGF also stimulated the mitogen activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and p38 (stress activated protein kinase-2), but not SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). Activation of p38 resulted in activation of MAP kinase activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). Inhibiting the VEGF-induced activation of ERK with PD098059 did not influence actin organization or cell migration but totally inhibited the VEGF-induced incorporation of thymidine into DNA. Inhibition of p38 activity by the specific inhibitor SB203580 led to an inhibition of HSP27 phosphorylation, actin reorganization and cell migration. The results indicate that the p38 pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration. By modulating cell migration, p38 may thus be an important regulator of angiogenesis.