Parkinson's disease patient-specific neuronal networks carrying the LRRK2 G2019S mutation unveil early functional alterations that predate neurodegeneration.

A deeper understanding of early disease mechanisms occurring in Parkinson's disease (PD) is needed to reveal restorative targets. Here we report that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) obtained from healthy individuals or patients harboring LRRK2 PD-causing mutation can create highly complex networks with evident signs of functional maturation over time. Compared to control neuronal networks, LRRK2 PD patients' networks displayed an elevated bursting behavior, in the absence of neurodegeneration. By combining functional calcium imaging, biophysical modeling, and DAn-lineage tracing, we found a decrease in DAn neurite density that triggered overall functional alterations in PD neuronal networks. Our data implicate early dysfunction as a prime focus that may contribute to the initiation of downstream degenerative pathways preceding DAn loss in PD, highlighting a potential window of opportunity for pre-symptomatic assessment of chronic degenerative diseases.

Generation of induced Pluripotent Stem Cells as disease modelling of NLSDM.

Neutral Lipid Storage Disease with Myopathy (NLSDM) is a rare defect of triacylglycerol metabolism, characterized by the abnormal storage of neutral lipid in organelles known as lipid droplets (LDs). The main clinical features are progressive myopathy and cardiomyopathy. The onset of NLSDM is caused by autosomal recessive mutations in the PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL). Despite its name, this enzyme is present in a wide variety of cell types and catalyzes the first step in triacylglycerol lipolysis and the release of fatty acids. Here, we report the derivation of NLSDM-induced pluripotent stem cells (NLSDM-iPSCs) from fibroblasts of two patients carrying different PNPLA2 mutations. The first patient was homozygous for the c.541delAC, while the second was homozygous for the c.662G>C mutation in the PNPLA2 gene. We verified that the two types of NLSDM-iPSCs possessed properties of embryonic-like stem cells and could differentiate into the three germ layers in vitro. Immunofluorescence analysis revealed that iPSCs had an abnormal accumulation of triglycerides in LDs, the hallmark of NLSDM. Furthermore, NLSDM-iPSCs were deficient in long chain fatty acid lipolysis, when subjected to a pulse chase experiment with oleic acid. Collectively, these results demonstrate that NLSDM-iPSCs are a promising in vitro model to investigate disease mechanisms and screen drug compounds for NLSDM, a rare disease with few therapeutic options.

Survival and its predictors from age 75 to 85 in men and women belonging to cohorts with marked survival differences to age 75: a comparative study in three Nordic populations.

BACKGROUND AND AIMS: While predictors of survival in older people have been examined in depth in a large number of studies, a literature search revealed no cross-national comparative prospective cohort studies on this issue. This study investigated survival and its predictors from age 75 to 85 among three local Nordic populations using survival data on national cohorts as background information. METHODS: The data were derived from national registers and from samples of 75-year old living in Denmark, Sweden, and Finland. The subjects were invited to take part in interviews and examinations focusing on different domains of health, functional capacity, and physical and social activities. RESULTS: The proportion of survivors to age 75 was markedly smaller among the Finnish men and women than Danish or Swedish subjects. In the local population no marked differences in survival from age 75 to 85 were observed between the groups of men, while women survived longer than men and longer in Göteborg than in Glostrup or Jyväskylä. Univariate models revealed 12 predictors of survival. In the multivariate models, the significant predictors among men related to physical fitness, whereas among women they pertained to social activities and morbidity. CONCLUSIONS: Despite great differences in the proportions of survivors to age 75, and excepting the survival advantage of women, only minor differences were present in the subjects' further survival to age 85. In the univariate analyses, many of the factors predictive of survival from age 75 to 85 were the same in the examined populations, whereas in the multivariate analyses differences between the sexes emerged.

Lower endothelial progenitor cell number, family history of cardiovascular disease and reduced HDL-cholesterol levels are associated with shorter leukocyte telomere length in healthy young adults.

BACKGROUND AND AIMS: Leukocyte telomere length (LTL) is a novel marker of cardiovascular (CV) risk. The aim of the study was to investigate the major determinants of LTL in a healthy young population at very low CV risk. METHODS AND RESULTS: LTL was determined in 82 healthy subjects (49M/33F; age37 ± 9yrs), normotensive and not taking any medication with different family history of cardiovascular disease (CVD) (24yes/58no). Fasting blood samples were drawn in all subjects for the determination of lipid profile, high sensitive C-reactive protein, uric acid, Plasminogen Activator Inhibitor-1 (PAI-1), LTL and Endothelial Progenitor Cell (EPC) number. LTL was assessed with a specific real-time PCR reaction in leukocyte DNA samples. LTL resulted inversely correlated with family history of CVD (t = 2.70; p = 0.009), age (r = -0.238; p = 0.032), waist circumference (r = -0.256; p = 0.02), triglycerides (r = -0.218; p = 0.049), PAI-1 (r = -0.288; p = 0.009) and directly correlated with HDL-cholesterol (r = 0.316; p = 0.004) and EPC number (r = 0.358; p = 0.002). At a multivariate analysis, family history of CVD (p = 0.013), EPC count (p = 0.003), and HDL-cholesterol (p = 0.017) were independently associated with LTL (r = 0.62). CONCLUSION: LTL is independently associated to CV risk factors also in healthy young adults.

Relationships between postural balance, rifle stability and shooting accuracy among novice rifle shooters.

The present study examined the relationships between shooting accuracy and shooters' behavioral performance, i.e., postural balance and gun barrel stability, among novice rifle shooters in intra- and inter-individual levels. Postural balance and rifle stability were assessed in terms of anteroposterior (VEL(AP)) and mediolateral (VEL(ML)) sway velocity of the movement of center of pressure, and horizontal (DEV(H)) and vertical (DEV(V)) deviation of the aiming point. The participants (n=58) performed 30 shots in the standing position at a distance of 10 m from the target. The data showed that shooting accuracy was related to postural balance and rifle stability, but only at the inter-individual level. The correlation coefficients between shooting score and behavioral performance variables ranged from -0.29 to -0.45. The stepwise multiple regression analysis revealed that the VEL(ML) and the DEV(H) as independent variables accounted for 26% of the variance in the shooting score. The results also suggested that postural balance is related to the shooting accuracy both directly and indirectly through rifle stability. As the role of postural balance appeared to be important in shooting performance, the use of additional balance training programs to improve a shooter's postural skills should be encouraged.

The predictive value of exercise testing for survival among 75-year-old men and women.

All 75-year-olds born in 1914 and living in the city of Jyväskylä, central Finland (n=388) were invited to study the predictive value of exercise test for mortality. Subjects who entered the laboratory (n=295) were to have a standard pre-test evaluation and perform a cycle ergometer exercise test. Subjects with complete background, exercise-test status and mortality data (n=282) were divided into three groups according to exercise-test status: a non-exercise test group (n=79), an exercise-test termination group (n=95), and an exercise-test completion group (n=108). Mortality was followed up for 9 years. The multivariate hazard ratio (HR) for death among the non-exercise test group compared with exercise-test completion group was 1.87 (CI 1.19-2.94). The multivariate HR for death among the exercise-test termination group compared with the exercise-test completion group was 0.95 (CI 0.58-1.54). High cycling power (W/kg body weight) in the exercise-test completion group was associated with a decreased risk for death with a multivariate HR 0.14 (CI 0.05-0.38). Performing an exercise test serves information on the risk of death that is incremental to clinical data and traditional risk factors of death in elderly people.

Postural balance in a random sample of 7,979 subjects aged 30 years and over.

BACKGROUND: Reliable normative data for force platform measurements of postural balance have not been available. METHODS: Data on postural balance were collected from a representative nationwide sample of a Finnish population aged >or=30 years (n = 7,979). As part of a comprehensive health survey (Health 2000), postural balance was measured with the help of a force platform system in four test conditions: normal standing with eyes open and closed (both for 30 s), semi-tandem (20 s) and tandem stand with eyes open (20 s). In addition, balance abilities were also evaluated by a non-instrumented field test. RESULTS: The main findings of this study indicated that the differences in balance between subjects belonging to different age categories were apparent already among young and middle-aged subjects. This is true, however, only for the more accurate force platform measurements, as the field test showed a clear ceiling effect up to 60 years of age. At higher ages both methods indicated a further, accelerating decline in balance function. In most cases, males tended to have more pronounced sway, as indicated by the speed and amplitude aspects of the movement of the center of pressure during the force platform registrations and these differences were larger in the older age groups. In contrast, in the field test a larger proportion of males were able to achieve the highest category (10 s in tandem stand) and the proportion of subjects unable to stand for a minimum of 10 s feet side by side was larger among females than males. These observations may partly be due to differences in the participation/acceptable performance in the different tests. In addition, the field test and force platform measurements may partially reflect different aspects of balance abilities. CONCLUSION: The results of the present study provide normative values for force platform balance tests at an age of 30 years and above. Deterioration in balance function clearly starts at relatively young ages and further accelerates from at about 60 years upwards. Due to systematic differences between males and females, separate normative values for both sexes are needed. Due to marked ceiling effects the field test can only be recommended for older individuals, aged >/=60. On the other hand, force platform registrations in the more demanding tests (semi-tandem and tandem stands) suffer from floor effects in the oldest age groups.

Optoelectronic measures in the analysis of running target shooting.

In this study, we examined the construct validity and practical significance of an optoelectronic shooting training system (Noptel ST 2000 Sport) for the technical analysis of running target shooting. A total of 37 male shooters of three different skill levels participated in the study. Principal component analysis revealed four common factors of 16 variables describing the aiming trajectory of the rifle barrel: (i) Holding area, (ii) Accuracy of aiming, (iii) Cleanness of triggering and (iv) Time on target. These factors were suggested to describe the essential components of running target shooting. According to the discriminant analysis, the shooters of various skill levels seemed to discriminate successfully into three groups when the aiming trajectory data were analysed. Finally, the aiming trajectory variables represented a 43% of the total variance in the shooting score. In summary, the present data indicated that the optoelectronic shooting training system had practical significance and supported the technical analysis of rifle barrel movement in running target shooting. A sub-set of variables, which reflect the essential information of running target shooting performance, were outlined for training and coaching purposes.

Comparison of Kistler 9861A force platform and Chattecx Balance System for measurement of postural sway: correlation and test-retest reliability.

Measurement of postural sway has several potential applications in sports medicine. Traditionally, however, rather complicated equipment has been applied. The purpose of the study was to compare two devices based on two different methods of measuring postural sway: (i) a sophisticated Kistler 9861A force platform (KIS)--which all but requires a laboratory setting--and (ii) Chattecx Balance System (CBS)--which is particularly suited for measurement of sway in the dynamic environment of sports. Measurement of sway was performed in 29 subjects twice at baseline and twice at follow-up 2-4 weeks later. One measurement consisted of four 25-second sequences (eyes open, parallel feet; eyes closed, parallel feet; eyes open, tandem Romberg; eyes closed, tandem Romberg). Factor analysis revealed influence of sight and stance on sway, marked interaction between these two factors and a decrease in sway on retest on the same day. Coefficients of variation were--KIS: 0.13-0.23; CBS: 0.11-0.25. Body height was a covariate for all parameters. Kistler 9861A force platform and CBS were correlated (baseline: r(s) = 0.47; follow-up: r(s) = 0.9). These findings suggest that, when the effects of acclimatization and covariance of body height were taken into account, CBS was as reliable and reproducible as KIS in our laboratory.

Body composition of 80-years old men and women and its relation to muscle strength, physical activity and functional ability.

OBJECTIVE: To measure body composition and analyse the relation to muscle strength, physical activity and functional ability in healthy, old subjects, and to relate the results to an optimal BMI level for the elderly. SETTING: Subjects aged 80 years living at home from the 1914-population in Glostrup, Denmark. SUBJECTS AND METHOD: 121 men and 113 women had their height and weight measured. Body fat mass and fat-free mass were assessed by bioelectrical impedance. Muscle strength was measured as handgrip, elbow flexion, knee extension, body flexion and body extension. Physical activity was self reported and functional ability was assessed by the Physical Performance Test (PPT) and self reported mobility including information about tiredness and help. RESULTS: After dividing BMI into three groups: BMI < 24, BMI 24-29 and BMI > 29 no relationship was seen between a BMI interval of 24-29 kg/m2, and physical activity and functional ability. BMI was related to body fat mass, and FFM was related to muscle strength. Muscle strength was related to mobility and PPT. Mobility and PPT were mutually related and were related to physical activity. CONCLUSION: Our cross sectional study did not support newly proposed guidelines for the elderly of an optimal BMI interval of 24-29 kg/m2. We found relations between body composition, muscle strength, physical activity and functional ability.

Paracrine and autocrine effects of fibroblast growth factor-4 in endothelial cells.

Recombinant Fibroblast Growth Factor-4 (FGF4) and FGF2 induce extracellular signal-regulated kinase-1/2 activation and DNA synthesis in murine aortic endothelial (MAE) cells. These cells co-express the IIIc/Ig-3 loops and the novel glycosaminoglycan-modified IIIc/Ig-2 loops isoforms of FGF receptor-2 (FGFR2). The affinity of FGF4/FGFR2 interaction is 20-30 times lower than that of FGF2 and is enhanced by heparin. Overexpression of FGF2 or FGF4 cDNA in MAE cells results in a transformed phenotype and increased proliferative capacity, more evident for FGF2 than FGF4 transfectants. Both transfectants induce angiogenesis when applied on the top of the chick embryo chorioallantoic membrane. However, in contrast with FGF2-transfected cells, FGF4 transfectants show a limited capacity to growth under anchorage-independent conditions and lack the ability to invade 3D fibrin gel and to undergo morphogenesis in vitro. Also, they fail to induce hemangiomas when injected into the allantoic sac of the chick embryo. In conclusion, although exogenous FGF2 and FGF4 exert a similar response in MAE cells, significant differences are observed in the biological behavior of FGF4 versus FGF2 transfectants, indicating that the expression of the various members of the FGF family can differently affect the behavior of endothelial cells and, possibly, of other cell types, including tumor cells.

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

Changes in bicycle ergometer test performance and survival in men and women from 50 to 60 and from 70 to 80 years of age: two longitudinal studies in the Glostrup (Denmark) population.

BACKGROUND: During the aging process longitudinal changes for sustained work in humans are poorly understood. Only a few longitudinal follow-up studies have been published thus far, and most of them have been based on highly selected groups, such as physical education teachers or senior athletes. OBJECTIVE AND METHODS: On the basis of random samples of a general population, the purpose of this study was to analyze changes in bicycle test performance in two longitudinal designs: a 10-year follow-up period from 50 to 60 years of age, using a submaximal test protocol, and another 10-year follow-up period from 70 to 80 years of age, using indirect voluntary maximal tests. In addition, the preventive value of the bicycle test results for survival at different age levels was analyzed. The subjects in the first part of the study were members of a random sample of originally 514 men and 461 women living in the Glostrup area, close to Copenhagen, Denmark, in 1964. Of these, 367 men (71.4%) and 206 women (44.7%) were tested at the age of 50 years, and 309 men and 245 women were tested 10 years later. The subjects in the second part of the study came from the same original sample. At 70 years of age 171 men and 154 women and at 80 years of age 70 men and 68 women took part in the maximal test. RESULTS: The submaximal test results between the ages of 50 and 60 years showed a mean annual decline in body mass adjusted maximal power in sustained work (W/kg) of 0.54% in men and of 0.90% in women. Between the ages of 70 and 80 years, when the indirect maximal tests were applied, the annual decline in men was on average 1.79% and in women 3.03%. When the associations of submaximal test results at ages 50-60 years and the voluntary maximal test results at the higher ages were analyzed, a moderate positive correlation was observed with the results obtained at the age of 70 years. The survival analyses showed that the submaximal bicycle test results (W/kg body mass) at the age of 60 years had a predictive value for survival in women during the subsequent 10-year period. The same was true for the maximal test results obtained at the age of 70 years in men; a significantly larger proportion of men in the lowest quintile died during the subsequent 10 years than of those belonging to the higher quintiles. CONCLUSIONS: The changes in body mass related maximal power in sustained work observed in this population on the basis of longitudinal studies among the age groups 50-60 and 70-80 years indicated a steeper decline at the higher ages. The decline was relatively more pronounced in women than in men. However, differences in the test protocols employed at different times limit the possibilities for overall comparisons across the data. The results of the submaximal bicycle ergometer tests in middle-aged female subjects (60 years old) had a predictive value for survival over the 10 years immediately following the test; likewise, the voluntary maximal test results at higher ages predicted survival in men.

Effects of 12-week shooting training and mode of feedback on shooting scores among novice shooters.

The mode of feedback has been shown to have an effect on motor skill learning. This study investigated effects of an intensive 12-week shooting training period, and of the mode of feedback on scores in standing and running target shooting among 30 novices with limited shooting experience. They were divided into one control (no training) and three training groups. One out of the three training groups received only KR (knowledge of results) while the two other groups received KR+KP (knowledge of performance). One of the KR+KP groups received additional visual feedback (FB-II group), which included videos, graphic and written materials about kinematic and kinetic characteristics of their respective shooting techniques compared to performance of international level shooters. Each training group improved their scores in the running target shooting. There were no significant differences between the three training groups in improvements of sum scores (performance outcome). The current study showed that among novice shooters both KR alone and KR+KP improved running target shooting outcome, and that KR and KP together did not lead to a significantly better shooting performance than KR alone.

Cardiac adverse effects and acute exercise in elderly subjects.

Cardiac arrhythmias, ST segment depressions and other cardiac adverse effects are occasionally seen in relation to physical exercise in elderly people. However, the magnitude, quality, and time-dependence of these events need to be clarified. During voluntary maximal cycle ergometer exercise (CEE), immediate cardiac disturbances as a reason for exercise termination were registered in a sample of 75-year-old men and women (N=203) (population group, PG). Any referrals to hospital following cardiac incidents within 24 hours post-CEE were obtained from patient records. Ambulatory ECG was recorded 24 hours before and after CEE in 23 elderly subjects (12 men, 11 women) capable of maximal effort (Holter group, HG). In the PG, 23.4% of the men and 6.4% of the women had to terminate CEE because of cardiac disturbances. One subject was referred to hospital because of exercise-induced atrial fibrillation. In the HG, no long-lasting effect of exercise on the occurrence of cardiac disturbances was found in elderly subjects capable of maximal effort.

Examining new models for the study of autocrine and paracrine mechanisms of angiogenesis through FGF2-transfected endothelial and tumour cells.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumour neovascularization. Several growth factors and cytokines have been shown to stimulate endothelial cell proliferation in vitro and in vivo and among them FGF2 was one of the first to be characterised. FGF2 is a Mr 18,000 heparin-binding cationic polypeptide that induces proliferation, migration, and protease production in endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells through two distinct classes of receptors, the high affinity tyrosine-kinase receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix. Besides experimental evidence for paracrine mode of action for FGF2, some observations raise the hypothesis that FGF2 may also play an autocrine role in endothelial cells. FGF2 may therefore represent a target for anti-angiogenic therapies. In order to assess the angiostatic potential of different classes of compounds, novel experimental models have been developed based on the autocrine and/or the paracrine capacity of FGF2.

Modulation of fibroblast growth factor-2 receptor binding, signaling, and mitogenic activity by heparin-mimicking polysulfonated compounds.

Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth.

Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.

Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.

Different tyrosine autophosphorylation requirements in fibroblast growth factor receptor-1 mediate urokinase-type plasminogen activator induction and mitogenesis.

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1-4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1-5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1-3F) and FGFR1-Y463/583/585/766F mutant (FGFR1-4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1-3F, and FGFR1-4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

Alterations of blood vessel development by endothelial cells overexpressing fibroblast growth factor-2.

A close relationship exists between angiogenesis and the formation of vascular lesions. The development of the vascular system in the chick embryo chorioallantoic membrane (CAM) may thus represent a model to study the effects of the deregulation of endothelial cell behaviour. Alterations of the developing vascular tree of the CAM were observed after exposure to murine aortic endothelial (MAE) cells overexpressing human fibroblast growth factor-2 (FGF2) cDNA (pZipFGF2 MAE cells), or to their conditioned medium (CM). pZipFGF2 MAE cells injected into the allantoic sac or applied on to the CAM of day 8-9 chick embryos induce neovascularization and the appearance of haemangioma-like lesions. This activity was not prevented by anti-FGF2 antibodies. The CM from pZipFGF2 MAE cells was also active when adsorbed into a gelatin sponge and applied on to the CAM, both in the absence and in the presence of anti-FGF2 antibodies. No effects on vessel development were exerted by parental MAE cells, FGF2-transfected NIH 3T3 fibroblasts, or their conditioned media. In vitro, pZipFGF2 MAE cell CM caused parental MAE cells to invade fibrin gels and to undergo morphogenesis on Matrigel. This activity was not mimicked by recombinant FGF2 nor affected by anti-FGF2 antibodies, and depended on a M (r) approximately 45 000 heat-labile heparin-binding factor. Size exclusion chromatography of pZipFGF2 MAE cell CM demonstrated that the in vitro activity co-purified with an in vivo angiogenic capacity. Thus, FGF2 overexpression in mouse endothelial cells induces the production of an angiogenic activity distinct from FGF2, which may contribute to the genesis of angioproliferative lesions.