Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active (alpha and/or alpha') and two regulatory (beta) subunits. It has been suspected that, among numerous other cellular functions, CKII might play a role in the control of mitogenic signaling. To test for such a role and its mechanism in intact cells, monoclonal antibodies (mAbs) were generated against CKII beta using a recombinant protein containing amino acids <em>20</em>-<em>20</em>0 of human CKII beta. The CKII beta-specific mAb with the highest reactivity, mAb IVG6 (classified as IgG1 with kappa light chains), was purified to homogeneity. It recognized a CKII beta epitope comprising the amino acids 140-156, a basic and highly conserved region. In addition, polyclonal antibodies (pAbs) were raised and made monospecific by affinity purification. pAbs-mediated quantitative immunofluorescence microscopy of human IMR-90 <em>fibroblasts</em> and/or Western blots of cell fractions revealed (i) CKII beta was present in exponentially <em>growing</em> cells at a 2-3-fold higher level than in quiescent cells, (ii) CKII beta was localized predominantly in the nucleus of cells (3-15-fold cytoplasmic level depending on cellular state and assay used), and (iii) the nuclear/cytoplasmic ratio of CKII beta was higher by a <em>factor</em> of 2 in exponentially <em>growing</em> cells. Consequently, mitogenic stimulation of quiescent cells by fetal calf serum doubled the nuclear/cytoplasmic ratio of CKII beta. The increase occurred within the 1st h of stimulation. The translocation of CKII beta into the nucleus was inhibited when mAb IVG6 was injected into the cytoplasm at the time of mitogenic stimulation. This microinjection also significantly inhibited the cell proliferation. The data imply that cytoplasmic CKII participates in the transmission of mitogenic signals by translocation into the nucleus.

It is well established that certain chemotherapeutic agents have potent antiangiogenic properties which may be part of their antitumor activity. Temozolomide (TMZ) is a lipophilic methylating agent used in the therapy of malignant melanoma and other tumors. We sought to determine whether TMZ is capable of inhibiting angiogenesis or influencing endothelial function. We used the in vivo chorioallantoic membrane (CAM) assay, and HUVEC-based in vitro Matrigel, adhesion and proliferation assays to determine the antiangiogenic effects of different doses of TMZ. In the CAM assay, angiogenesis was significantly inhibited by 5 microM TMZ, a concentration also found to be effective in interfering with in vitro angiogenesis as measured by the Matrigel assay. For the inhibition of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-, vascular endothelial <em>growth</em> <em>factor</em> (VEGF)- or beta-phorbol 12-myristate-13-acetate (PMA)-induced endothelial cell proliferation or endothelial cell adhesion to fibronectin, TMZ concentrations of at least 25 microM were necessary, indicating that bFGF-, VEGF- or protein kinase C-mediated pathways may not primarily be involved in the observed antiangiogenic effect. Thus, we could demonstrate that TMZ inhibits angiogenesis at low, non-toxic doses that correspond to the plasma concentrations achieved by an oral application of <em>20</em> mg/m2 every 8 h. This 'metronomic' scheduling has already been used in phase I studies and has produced antitumor effects. Therefore, the antitumor activity of TMZ may, at least in part, be due to its antiangiogenic properties. The precise mechanism of its antiangiogenic action remains to be elucidated.

BACKGROUND

Fibroblasts have been universally recognised in tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. Recently, pirfenidone (PF) has been shown to both ameliorate progressive fibrosis and reduce established scarring after ureteric obstruction (UUO) in the rat, suggesting that it is a novel anti-fibrotic agent. The objective of this study was therefore to determine if these effects include down-regulation of fibroblast function.

METHODS

Cortical fibroblasts were obtained from outgrowth cultures of renal tissue isolated from kidneys 3 days after UUO and constituted 100% of cells studied. Functional studies examined the effects of 20 and 200 microg/ml PF on basal serum stimulated activity. Activation was examined by western blotting for alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Cell proliferation, collagenase activity and collagen production were determined from kinetic studies, zymography for MMP2 and [3H] proline incorporation in collagenous proteins respectively.

RESULTS

Proliferation, as measured by [3H] thymidine incorporation, was reduced in dose dependent manner by 20 and 200 microg/ml PF (p<0.05; 200 vs 0 microg/ml). Likewise, 200 microg/ml PF reduced cell population growth over 5 days of culture (p<0.05 vs 0 microg/ml). PF (200 microg/ml) decreased alphaSMA and CTGF protein expression to 66+/-13 and 37+/-26% of basal levels respectively (both p<0.05 vs 0 microg/ml). Synthesis of collagen was unaffected by PF. Maximal dose of PF produced a modest reduction in MMP2 lytic activity (p=0.05). Effects of PF were independent of cell toxicity.

CONCLUSIONS

Down-regulation of renal fibroblast activation and proliferation are specific actions of PF.

BACKGROUND

The role of bevacizumab, a recombinant humanized monoclonal antibody directed against vascular endothelial growth factor, in the treatment of pancreatic cancer remains unclear. The objectives of this study were to determine safety and efficacy in chemotherapy-naive patients with metastatic pancreatic cancer receiving bevacizumab in combination with fixed-dose rate (FDR) gemcitabine and low-dose cisplatin.

METHODS

Eligible patients received gemcitabine 1,000 mg/m2 at FDR infusion (10 mg/m(2) per minute), cisplatin 20 mg/m(2), and bevacizumab 10 mg/kg, on days 1 and 15 of a 28-day cycle. Patients were monitored by computed tomography scans every two cycles and monthly serum CA19-9 measurements.

RESULTS

Of 52 patients eligible for analysis, ten (19.2%) had an unconfirmed response and 30 (57.7%) had stable disease. Of 35 patients with elevated baseline CA19-9 levels, 20 (57.1%) had>> or = 50% biomarker decline during treatment. Median time to tumor progression was 6.6 months and median survival was 8.2 months (estimated 1-year survival, 36%). Grade 3/4 toxicities possibly related to bevacizumab included thromboembolic events (15.1%), hypertension (13.2%), gastrointestinal bleeding (9.4%), cardiac events (7.5%), and bowel perforation (5.7%). Plasma vascular endothelial growth factor and basic fibroblast growth factor levels and circulating tumor cell concentration did not correlate with overall survival, either at baseline or after 2 months of therapy.

CONCLUSIONS

This bevacizumab-containing study regimen is modestly effective in patients with metastatic pancreatic cancer, although occasional serious complications may occur. Given the negative results of CALGB 80303, future efforts should be focused on identifying those specific patients who are most likely to benefit from bevacizumab-based therapy.

We investigated the distribution of alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin isoforms in human heart during development, hypertrophy, and failure. At <em>20</em> weeks of fetal life, alpha-skeletal actin was localized in a small proportion of subendocardial and papillary muscle cardiomyocytes. At this gestation time, diffuse alpha-cardiac actin staining was observed, associated with focal expression of alpha-smooth muscle actin. In normal adult subjects, alpha-skeletal actin positive cardiomyocytes were distributed in a transmural gradient with the highest proportion located subendocardially. In myocardial hypertrophy and cardiomyopathies, the amount of alpha-skeletal actin was increased and diffuse staining was seen in all layers of ventricular myocardium, with the exception of idiopathic dilated cardiomyopathies. Cardiomyocytes were negative for alpha-smooth muscle actin in all pathological situations studied. As expected, <em>fibroblasts</em> in post-infarct scars expressed alpha-smooth muscle actin and transforming <em>growth</em> <em>factor</em>-beta1 but, surprisingly, were negative for these proteins in interstitial fibrosis. Our results demonstrate that increased expression of alpha-skeletal actin in the diseased human heart is associated with increased myocyte stretch, increased wall stress, and pressure overload, but not with idiopathic dilated cardiomyopathies. They also suggest that fibrotic changes develop with different mechanisms in scars versus interstitial fibrosis.

The potential <em>growth</em> stimulating effects of the blow fly, Phaenicia sericata, on mammalian tissue were assessed by exposing human <em>fibroblast</em> tissue culture to maggot extracts. The <em>growth</em> effects of these extracts were compared to those of epidermal <em>growth</em> <em>factor</em> (EGF), recombinant interleukin 6 (IL6), and the insect hormone <em>20</em>-hydroxyecdysone (EC). Results of dose-response experiments revealed that EGF had a maximum <em>fibroblast</em> stimulation at 66078 +/- 1979 counts per minute (cpm), with peak counts on day 6 of culture, as measured by [3H]-thymidine incorporation. P. sericata hemolymph (HL) and alimentary secretions (AS) and EC were also demonstrated to stimulate resting <em>fibroblast</em> tissue cultures, but the maximal stimulations only achieved 12% of EGF. Their <em>growth</em> rates plateaued between days 4 and 6. Addition of both HL and AS, as well as EC, significantly increased the <em>growth</em> rate of EGF-stimulated <em>fibroblasts</em>; AS increased the maximal stimulation of IL6-stimulated <em>fibroblasts</em>. These studies suggest the existence of intrinsic <em>factors</em> within the maggot which may be responsible for the <em>growth</em>-stimulating effects seen in maggot-infested wounds.

BACKGROUND

Fibroblast growth factor-23 (FGF-23) is a phosphate regulatory hormone that directly stimulates left ventricular hypertrophy in experimental models. The role of FGF-23 in cardiovascular disease development in the general population is unclear. We tested associations of FGF-23 with major subclinical and clinical cardiovascular disease outcomes in a large prospective cohort.

RESULTS

We evaluated 6547 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) who were initially free of cardiovascular disease. We measured serum FGF-23 using the Kainos immunoassay. The MESA measured left ventricular mass by MRI, coronary calcium by computed tomography, and carotid intima-media thickness by ultrasound. The MESA adjudicated incident heart failure, coronary heart disease, and stroke by medical record review. After adjustment, the highest FGF-23 quartile was associated with an estimated 2.4-g greater left ventricular mass (95% confidence interval, 0.4-4.5 greater) and a 26% greater odds of higher coronary calcium scores (95% confidence interval, 9%-46% greater) compared with the lowest quartile. During 7.5-year follow-up, each 20-pg/mL higher FGF-23 concentration was associated with a 19% greater risk of heart failure (95% confidence interval, 3%-37% greater) and a 14% greater risk of coronary heart disease (95% confidence interval, 1%-28% greater). FGF-23 was not associated with carotid intima-media thickness or stroke.

CONCLUSIONS

Higher serum FGF-23 concentrations are associated with subclinical cardiac disease and with new heart failure and coronary disease events, but not with carotid intima-media thickness or stroke. FGF-23 may be a novel cardiovascular risk factor in the general population.

Many mitogens cause rapid changes in intracellular pH and Ca2+. We studied the patterns of pH and Ca2+ changes after exposure of murine <em>fibroblasts</em> to platelet-derived <em>growth</em> <em>factor</em> (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca2+ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. PDGF and bombesin caused a rapid (maximum change, less than 2 min) cytoplasmic acidification of 0.03 pH unit followed by a slower (5-10 min) alkalinization of approximately 0.11 pH unit above the resting pH of 6.88. PMA caused alkalinization without causing the early acidification. Bradykinin caused rapid acidification without the slower net alkalinization. Ionomycin also caused acidification without subsequent alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca2+ response were also determined for each agent. PDGF and bombesin caused a transient increase in cytoplasmic Ca2+ from a resting level of 85 +/- 12 nM to 190 +/- 12 nM within 2 min and return to baseline within 5 min. PMA caused no change in intracellular Ca2+. Bradykinin caused the most rapid (maximum response, less than <em>20</em> sec) increase in intracellular Ca2+. For each agonist, the Ca2+ transient could be blocked by buffering intracellular Ca2+ with quin-2. In Ca2+-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. We propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca2+. However, <em>growth</em> <em>factor</em>-induced alkalinization via the Na+/H+ exchanger is independent of changes in Ca2+.

<em>Growth</em> <em>factor</em> activity was partially purified from human renal tumors and a human bladder cancer cell line by heparin-Sepharose chromatography. This activity stimulated bovine capillary endothelial cell proliferation and DNA synthesis in BALB/c 3T3 cells. Partially purified <em>growth</em> <em>factor</em> preparations from these tumors contained a protein with an approximate molecular weight of 17,000 which was recognized by a polyclonal antiserum raised against a peptide fragment of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). This <em>growth</em> <em>factor</em> activity appears to be related to basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Measurement of FGF-like activity in 50 urine samples from 32 adult males showed that 55% (6 of 11) of the urine samples from patients with bladder cancer and 100% (7 of 7) of the urine samples from patients with kidney cancer contained activity equivalent to more than <em>20</em> ng of basic FGF/h of urine production. In contrast, only 6% (2 of 32) of the urine samples from controls, patients with a benign disease, or patients with a history of bladder or kidney cancer contained this level of <em>growth</em> <em>factor</em> activity. These results suggest that patients with bladder or kidney cancer release an FGF-like <em>factor</em> into urine which may be used as a marker for these tumors.

OBJECTIVE

Cardiac mast cells participate in myocardial dysfunction, but the mechanisms are presently unknown.

METHODS

By examining spontaneously hypertensive rats (SHRs) during their entire lifespan, we attempted to define the role of mast cells in the induction of cardiac hypertrophy and transition to heart failure.

RESULTS

By contrast to normotensive littermates, hearts of newborn SHRs already contained mast cells. In the prehypertensive (2-week-old) SHRs, the increased expression of c-kit and soluble stem cell <em>factor</em> correlated with an increased number of cardiac mast cells. The mast cells contained tumour necrosis <em>factor</em>-alpha which, together with nuclear <em>factor</em> kappa-B (NF-kappaB) and interleukin (IL)-6, was significantly induced in the prehypertensive SHRs. Stimulation of cardiac mast cells with compound 48/80 in an ex-vivo Langendorff heart perfusion system resulted in increased expression of nuclear <em>factor</em> Kappa-B (NF-kappaB) (four-fold) and IL-6 (nine-fold) mRNA in the left ventricles of adult rat hearts. In the presence of an inhibitor of mast cell degranulation, disodium cromoglycate, the induced expression of NF-kappaB and IL-6 was inhibited. In the late hypertensive stage, the hearts of SHRs with advanced cardiac hypertrophy (12-month-old) and heart failure (<em>20</em>-month-old) had significantly increased levels of transforming <em>growth</em> <em>factor</em> (TGF)-beta1 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and displayed increased myocardial fibrosis. Activated mast cells were a major source of TGF-beta1 and bFGF, and localized to areas of myocardial fibrosis.

CONCLUSIONS

By synthesizing and secreting prohypertrophic cytokines and profibrotic growth factors, cardiac mast cells participate in the induction of cardiac hypertrophy and cardiac fibrosis, which are the key steps in the transition to heart failure.

Addition of various heparinoids to the lactose-introduced, water-soluble chitosan (CH-LA) aqueous solution produces an injectable chitosan/heparinoid hydrogel. In the present work, we examined the capability of the chitosan/non-anticoagulant heparin (periodate-oxidized (IO(4)-) heparin) hydrogel to immobilize <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2, as well as the controlled release of FGF-2 molecules from the hydrogel in vitro and in vivo. The hydrogel was biodegraded in about <em>20</em> days after subcutaneous injection into the back of a mouse. When the FGF-2-incorporated hydrogel was subcutaneously injected into the back of both mice and rats, a significant neovascularization and fibrous tissue formation were induced near the injected site. These results indicate that the controlled release of biologically active FGF-2 molecules is caused by biodegradation of the hydrogel, and that subsequent induction of the vascularization occurs.

Human meniscus cells have a predominantly fibrogenic pattern of gene expression, but like chondrocytes they proliferate in monolayer culture and lose the expression of type II collagen. We have investigated the potential of human meniscus cells, which were expanded with or without <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), to produce matrix in three-dimensional cell aggregate cultures with a chondrogenic medium at low (5%) and normal (<em>20</em>%) oxygen tension. The presence of FGF2 during the expansion of meniscus cells enhanced the re-expression of type II collagen <em>20</em>0-fold in subsequent three-dimensional cell aggregate cultures. This was increased further (400-fold) by culture in 5% oxygen. Cell aggregates of FGF2-expanded meniscus cells accumulated more proteoglycan (total glycosaminoglycan) over 14 days and deposited a collagen II-rich matrix. The gene expression of matrix-associated proteoglycans (biglycan and fibromodulin) was also increased by FGF2 and hypoxia. Meniscus cells after expansion in monolayer can therefore respond to chondrogenic signals, and this is enhanced by FGF2 during expansion and low oxygen tension during aggregate cultures.

Wild-type p53 is necessary for the <em>growth</em> arrest of human diploid <em>fibroblasts</em> (HDF) (and many other cell types) at the end of their proliferative lifespan. Although p53 may actively mediate senescence, possibly in response to telomere erosion, it is however equally possible that it is merely a permissive <em>factor</em> required for response to some other inducer. To address this question, we have generated stable transfectants of early passage HDF, represented here by clone LacZ21, in which expression of a beta-galactosidase reporter construct reflects p53 transactivation activity. During continuous passage, the proportion of beta-gal positive LacZ21 cells remained below 2% for 25 population doublings (pd), first became significantly increased after 29 pd, and thereafter increased rapidly, reaching a maximum of 88% in fully-senescent cells (32 pd), which exceeded the response observed following an optimum dose (<em>20</em> J/m2) of u.v. radiation. Correspondingly, the proportion of cells incorporating bromodeoxyuridine (BrdU) (initially 45-50%) began to fall at 29 pd and thereafter dropped rapidly to below 1% by pd 32. There was therefore a near-perfect reciprocal relationship between reporter construct expression and DNA synthesis as cells approached senescence. Furthermore, a dominant-negative p53 mutant (introduced by retroviral transduction) rescued LacZ21 cells from senescence and generated colonies with extended lifespan in which beta-gal expression was totally abolished. These data, although not excluding the need for other p53 functions, strongly suggest that p53-mediated transactivation of <em>growth</em> regulatory genes is a direct trigger, rather than a permissive <em>factor</em>, for cellular senescence.

The functional significance of coronary collaterals in humans has been debated for many years. Correlations have now been made between the anatomic appearance of coronary collateral vessels visualized at the time of intracoronary thrombolytic therapy during the acute phase of myocardial infarction and the creatine kinase time--activity curve, infarct size, and aneurysm formation. These studies demonstrate a protective role of collaterals in hearts with coronary obstructive disease, showing smaller infarcts, less aneurysm formation, and improved ventricular function compared with patients in whom collaterals were not visualized. There is ample evidence that collaterals respond to myocardial ischemia by opening preexistent channels. When the cardiac myocyte is rendered ischemic, collaterals develop actively by <em>growth</em> with DNA replication and mitosis of endothelial and smooth muscle cells. Heparin-binding <em>growth</em> <em>factors</em> are present in the heart, but their biological activity is quiescent under normal physiological conditions. Once ischemia develops, these <em>factors</em> are activated and become available for receptor occupation, which may initiate angiogenesis after exposure to exogenous heparin. This characteristic of heparin to potentiate the mitogenic activity of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> has recently been used in the clinical setting as a possible therapeutic modality in patients with coronary artery disease. Patients performing <em>20</em> rounds of exercise serially after receiving intravenous injection of heparin showed significantly greater increases in exercise capacity and improvement of clinical symptoms compared with the control group who performed the same exercise without heparin. Further study of neovascularization may lead to a new therapeutic strategy for ischemic heart disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Degeneration of dopaminergic neurons of the substantia nigra causes Parkinson's disease. Therefore, neurotrophic <em>factors</em> for dopaminergic neurons are of substantial clinical interest. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>20</em> preferentially expressed in the substantia nigra pars compacta (SNPC) of the rat brain significantly enhanced the survival of midbrain dopaminergic neurons. Here we examined the mechanism of action of FGF-<em>20</em> on dopaminergic neurons. FGF-<em>20</em> slightly enhanced the survival of total neurons of the midbrain, indicating that it preferentially enhanced the survival of dopaminergic neurons. FGF receptor (FGFR)-1c was found to be expressed abundantly in dopaminergic neurons in the SNPC but at much lower levels in neurons of other midbrain regions by in situ hybridization. FGF-<em>20</em> was also found to bind FGFR-1c with high affinity with the BIAcore system. Furthermore, FGF-<em>20</em> activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGFs. Both the FGFR-1 inhibitor SU5402 and the MAPK pathway inhibitor PD98059 also significantly inhibited the activation of the MAPK pathway by FGF-<em>20</em> and the neurotrophic activity of FGF-<em>20</em>. The present findings indicate that the activation of the MAPK pathway by FGF-<em>20</em> signaling through FGFR-1c plays important roles in the survival of dopaminergic neurons in the SNPC.

Plaque vascularity has been implicated in its <em>growth</em> and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque <em>growth</em>. To inhibit <em>growth</em> of vasa vasorum in atherogenic mice and assess its effect on plaque <em>growth</em>, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-1(23), that has significant antiangiogenic activity. Female LDLR(-/-)ApoB-48-deficient mice fed Paigen's diet without cholate for <em>20</em> weeks received rPAI-1(23) treatment (n=21) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen's diet and treated with saline for the last 6 weeks (n=16) and mice fed Paigen's diet until the onset of treatment (n=14). The rPAI-1(23) treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-1(23) treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-1(23)-treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 expression, which is inhibited by rPAI-1(23). In conclusion, rPAI-1(23) inhibits <em>growth</em> of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, leading to reduced plaque <em>growth</em> in atherogenic female LDLR(-/-)ApoB-48-deficient mice.

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of <em>fibroblasts</em>. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 <em>fibroblast</em> cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (<em>20</em>-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating <em>factor</em> of PDGF-mediated tyrosine kinase signaling.

A cell-free assay has been developed to detect and characterize a nerve <em>growth</em> <em>factor</em> (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-<em>20</em>-fold; activation is nearly as high with <em>fibroblast</em> <em>growth</em> <em>factor</em>, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal <em>growth</em> <em>factor</em> and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for <em>growth</em>-<em>factor</em>-promoted neuronal differentiation.

Epidermal <em>growth</em> <em>factor</em> (EGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and nerve <em>growth</em> <em>factor</em> (NGF), which stimulate the phosphorylation of proteins on tyrosine in PC12 cells, initiate these modifications through ligand-specific cell surface receptors that contain the causative tyrosine kinases. One apparent substrate for these enzymes is phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates the D-3 position of the inositol ring and associates with several protein tyrosine kinases, as indicated by the fact that it is immunoprecipitated from EGF-, bFGF-, and NGF-stimulated PC12 cells by an anti-phosphotyrosine antibody. All three <em>growth</em> <em>factors</em> increase immunoprecipitable PI 3-kinase activity after 2 min of addition at concentrations able to stimulate either mitogenic or neurotrophic responses in PC12 cells. The level of stimulation of PI 3-kinase activity by EGF, bFGF, and NGF is 15- to <em>20</em>-fold, 2- to 3-fold, and 8- to 10-fold, respectively. Moreover, tyrosine phosphorylation of PI 3-kinase was detected in EGF-, bFGF-, and NGF-stimulated PC12 cells, and the amount of the phosphorylation correlated with the level of stimulation of enzyme activity. In contrast, phosphatidylinositol 4-kinase, which produces the inositol phospholipids cleaved by phospholipase C-gamma to yield diacylglycerol and inositol-1,4,5-trisphosphate, is not affected by these <em>growth</em> <em>factors</em>. The pattern of stimulation of PI 3-kinase does not correlate with the induction of neurite out<em>growth</em> but rather with the mitotic responses, suggesting that PI 3-kinase and its products may be more important for signaling in cell division than in trophic processes. However, the levels of phosphatidylinositol 3-phosphate do not coincide with the stimulation of [3H]thymidine incorporation by these <em>growth</em> <em>factors</em>, rendering its role in mitotic functions, at least in PC12 cells, also uncertain.

OBJECTIVE

Fibrotic sequelae remain the most important dose-limiting toxicity of radiation therapy to soft tissue. Functionally, this is reflected in loss of range of motion and muscle strength and the development of limb edema and pain. Tumor necrosis factor alpha and fibroblast growth factor 2 (FGF2), which are abnormally elevated in irradiated tissues, may mediate radiation fibrovascular injury.

METHODS

In an open label drug trial, we studied the effects of pentoxifylline (400 mg orally tid for 8 weeks) on 30 patients who displayed late, radiation-induced fibrosis at 1 to 29 years posttreatment (40 to 84 Gy). The primary outcome measurement was change in physical impairments thought to be secondary to radiation, including active and passive range of motion (AROM and PROM), muscle strength, limb edema, and pain. Plasma levels of cytokines (tumor necrosis factor alpha and FGF2) also were measured. Twenty-seven patients completed baseline and 8-week assessments, and 24 patients completed baseline, 8-week, and 16-week assessments.

RESULTS

After 8 weeks of pentoxifylline intervention, 20 of 23 patients with impaired AROM and 19 of 22 with impaired PROM improved; 11 of 19 patients with muscle weakness showed improved motor strength; five of seven patients with edema had decreased limb girth; and nine of 20 patients had decreased pain. Pretreatment FGF2 levels dropped from an average of 44.9 pg/mL to 24.0 pg/mL after 8 weeks of treatment.

CONCLUSIONS

Patients receiving pentoxifylline demonstrated improved AROM, PROM, and muscle strength and decreased limb edema and pain. Reversal of these delayed radiation effects was associated with a decrease in circulating FGF2.

BACKGROUND

The platelet gel is made by embedding concentrate platelets within a semisolid (gel) network of polymerized fibrin. It is believed that this blood component will be used more and more in the treatment of several clinical conditions and as an adjunctive material in tissue engineering. Several systems are available to produce platelet-rich plasma (PRP) for topical therapy. Recently, a new system became commercially available, Plateltex. Here we report the technical performance of this system in comparison with the performance of other commercially available systems: PRGF, PRP-Landesber, Curasan, PCCS, Harvest, Vivostat, Regen and Fibrinet.

METHODS

Both the PRP and the gel were prepared according to the manufacturer's directions. The blood samples of <em>20</em> donors were used. The yield, the efficiency, and the amount of platelet-derived <em>growth</em> <em>factor</em> AB (PDGF-AB), transforming <em>growth</em> <em>factor</em> beta, vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em> were measured in the resulting PRP. The feature of the batroxobin-induced gelation was evaluated.

RESULTS

The yield, the collection efficiency and the growth factor content of Plateltex were comparable to those of most of the other available systems. The gelation time was not dependent on the fibrinogen concentration; however, it was strongly influenced by the contact surface area of the container where the clotting reaction took place (P < 0.0001).

CONCLUSIONS

Plateltex provided platelet recovery, collection efficiency and PDGF-AB availability close to those provided by other systems marketed with the same intended use. Batroxobin, the enzyme provided to induce gelation, acts differently from thrombin, which is used by most other systems. Platelets treated with thrombin become activated; they release their growth factors quickly. Furthermore, thrombin-platelet interaction is a physiological mechanism that hastens the clot-retraction rate. On the contrary, platelets treated with batroxobin do not become activated; they are passively entrapped within the fibrin network, and their growth factor release occurs slowly. In these conditions, the clot retraction takes longer to occur. According to these differences between thrombin and batroxobin, it is expected that batroxobin-induced PRP activation will tailor slow release of the platelet content, thus, providing longer in loco availability of trophic factors. In selected clinical conditions, this durable anabolic factor availability might be preferable to quick thrombin-induced growth factor release.

BACKGROUND

Angiogenesis is a target in the treatment of ovarian cancer. Nintedanib, an oral triple angiokinase inhibitor of VEGF receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, has shown activity in phase 2 trials in this setting. We investigated the combination of nintedanib with standard carboplatin and paclitaxel chemotherapy in patients with newly diagnosed advanced ovarian cancer.

METHODS

In this double-blind phase 3 trial, chemotherapy-naive patients (aged 18 years or older) with International Federation of Gynecology and Obstetrics (FIGO) IIB-IV ovarian cancer and upfront debulking surgery were stratified by postoperative resection status, FIGO stage, and planned carboplatin dose. Patients were randomly assigned (2:1) via an interactive voice or web-based response system to receive six cycles of carboplatin (AUC 5 mg/mL per min or 6 mg/mL per min) and paclitaxel (175 mg/m(2)) in addition to either 200 mg of nintedanib (nintedanib group) or placebo (placebo group) twice daily on days 2-21 of every 3-week cycle for up to 120 weeks. Patients, investigators, and independent radiological reviewers were masked to treatment allocation. The primary endpoint was investigator-assessed progression-free survival analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01015118.

RESULTS

Between Dec 9, 2009, and July 27, 2011, 1503 patients were screened and 1366 randomly assigned by nine study groups in 22 countries: 911 to the nintedanib group and 455 to the placebo group. 486 (53%) of 911 patients in the nintedanib group experienced disease progression or death compared with 266 (58%) of 455 in the placebo group. Median progression-free survival was significantly longer in the nintedanib group than in the placebo group (17·2 months [95% CI 16·6-19·9] vs 16·6 months [13·9-19·1]; hazard ratio 0·84 [95% CI 0·72-0·98]; p=0·024). The most common adverse events were gastrointestinal (diarrhoea: nintedanib group 191 [21%] of 902 grade 3 and three [<1%] grade 4 vs placebo group nine [2%] of 450 grade 3 only) and haematological (neutropenia: nintedanib group 180 [20%] grade 3 and 200 (22%) grade 4 vs placebo group 90 [20%] grade 3 and 72 [16%] grade 4; thrombocytopenia: 105 [12%] and 55 [6%] vs 21 [5%] and eight [2%]; anaemia: 108 [12%] and 13 [1%] vs 26 [6%] and five [1%]). Serious adverse events were reported in 376 (42%) of 902 patients in the nintedanib group and 155 (34%) of 450 in the placebo group. 29 (3%) of 902 patients in the nintedanib group experienced serious adverse events associated with death compared with 16 (4%) of 450 in the placebo group, including 12 (1%) in the nintedanib group and six (1%) in the placebo group with a malignant neoplasm progression classified as an adverse event by the investigator. Drug-related adverse events leading to death occurred in three patients in the nintedanib group (one without diagnosis of cause; one due to non-drug-related sepsis associated with drug-related diarrhoea and renal failure; and one due to peritonitis) and in one patient in the placebo group (cause unknown).

CONCLUSIONS

Nintedanib in combination with carboplatin and paclitaxel is an active first-line treatment that significantly increases progression-free survival for women with advanced ovarian cancer, but is associated with more gastrointestinal adverse events. Future studies should focus on improving patient selection and optimisation of tolerability.

BACKGROUND

Boehringer Ingelheim.

c-Src non-receptor tyrosine kinase is an important component of the platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in <em>fibroblasts</em>. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src-family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of <em>20</em> novel Src-family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src-family kinase substrates - Calpain 2, Eps15 and Trim28 - in a c-Src-dependent fashion.

Early molecular events during the development and regeneration of skin appendages were studied using cultured chicken skin explants with epithelial-mesenchymal recombination. The explant epithelium was separated from the mesenchyme, rotated 90 degrees or 180 degrees, recombined with the mesenchyme, and cultured. After this procedure, existing feather buds disappeared and new buds were regenerated. The location of the new buds is determined by the original dermal condensations, whereas the orientation is dictated by the original epithelium. The temporal expression of key morphogenetic molecules was examined 3, 6, and <em>20</em> h after recombination by whole-mount in situ hybridization and immunostaining. The results showed the following. (i) Placode formation and the expression of wingless-int (Wnt) 7a and Msx-1 in the placode epithelium are mesenchyme dependent. (ii) Hox C6 and neural cell adhesion molecule (NCAM) expression in the anterior mesenchyme is placode epithelium dependent. (iii) Bone morphogenetic protein (BMP)-2, BMP-4, and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-4 expression in the original dermal condensations was unaffected by recombination. (iv) Old dermal condensations can induce new placodes with new Wnt 7a, sonic hedgehog (Shh), and Msx-1 and -2 expression. (v) The new placode epithelium can then induce new Hox C6 and NCAM microgradients in the feather bud mesenchyme. (vi) The order of appearance can be classified into four groups in the following order: BMP-2, BMP-4, and FGF-4 (peptide <em>growth</em> <em>factors</em>); Wnt 7a and Shh (Drosophila segment polarity gene homologs); Msx-1 and Msx-2 (Msx class homeobox genes); and then Hox C6 (Hox class homeobox genes) and NCAM (adhesion molecules). These results suggest an order for the molecular cascade during the inductive phase of skin appendage development.