BACKGROUND

To examine the effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and transforming growth factor beta 2 (TGF(beta2)) on bovine retinal pigment epithelial (RPE) cell migration and proliferation.

METHODS

Cultured bovine RPE cells were treated with 10 ng/ml PDGF, bFGF, aFGF, IGF-1, EGF, or TGF(beta2). RPE cell migration studies were performed in multiwell plates confluently covered with RPE cells. After inhibition of proliferation and denudation of half of each well, cells were incubated with various growth factors. Migration was measured as the number of cells that had entered the denuded area after 20 h. RPE cell proliferation was determined by [(3)H]thymidine incorporation after growth factor stimulation for 24 h.

RESULTS

RPE cell migration was significantly enhanced after incubation with PDGF (stimulation of 213% compared to the negative control, p = 0.002), bFGF (206%, p = 0.003), aFGF (175%, p = 0.003), IGF-1 (150%, p = 0.003), and EGF (144%, p = 0.003). RPE cell proliferation was stimulated by bFGF (322% compared to the negative control, p < 0.005), PDGF (119%, p < 0.005), aFGF (121%, p < 0.005), and EGF (94%, p < 0.005). IGF-1 showed no significant effect on RPE cell proliferation; TGF(beta2) displayed no effect on RPE cell migration nor on proliferation.

CONCLUSIONS

The peptide growth factors PDGF, bFGF, aFGF, IGF-1, and EGF play an important role in initiating RPE cell migration. Basic FGF, PDGF, aFGF, and EGF stimulate RPE cell DNA synthesis.

BACKGROUND

Serum phosphorus levels have been associated with mortality in some but not all studies. Because dietary intake prior to measurement can affect serum phosphorus levels, we hypothesized that the association between serum phosphorus level and mortality is strongest in those who have fasted longer.

METHODS

Prospective cohort study.

METHODS

Nationally representative sample of 12,984 participants <em>20</em> years or older in the Third National Health and Nutrition Examination Survey (1988-1994).

METHODS

Serum phosphorus level, fasting duration (dichotomized as ≥ 12 or < 12 hours).

RESULTS

All-cause and cardiovascular mortality determined by death certificate data from the National Death Index.

METHODS

Serum phosphorus measured in a central laboratory and fasting duration recorded as time since food or drink other than water was consumed.

RESULTS

Individuals fasting 12 or more hours had lower serum phosphorus levels than those fasting less than 12 hours (3.34 vs 3.55 mg/dL; P < 0.001) and higher correlation with repeat measurement (0.66 vs 0.53; P = 0.002). In multivariable-adjusted Cox regression models, the highest quartile of serum phosphorus was associated with increased mortality in participants fasting 12 or more hours (adjusted HR, 1.74; 95% CI, 1.38-2.<em>20</em>; reference, lowest quartile) but not in participants fasting less than 12 hours (adjusted HR, 1.08; 95% CI, 0.89-1.32; P for interaction = 0.002). Relationships were consistent using 8 hours as the fasting cutoff point or cardiovascular mortality as the outcome.

CONCLUSIONS

Observational study, lack of fibroblast growth factor 23 or intact parathyroid hormone measurements.

CONCLUSIONS

Fasting but not nonfasting serum phosphorus levels were associated with increased mortality. Risk prognostication based on serum phosphorus may be improved using fasting levels.

The normal development of calvarial bones and sutures critically relies on proper signalling through Fgf receptors, but the source and identity of cognate ligands have remained unknown. Reverse transcriptase polymerase chain reaction analysis in this study shows that a broad range of Fgf ligands are expressed in the coronal sutures separating the parietal and frontal bones. Analysis by whole mount in situ hybridization further reveals distinct expression patterns for Fgf-18, Fgf-<em>20</em>, and by comparison, Fgf-9, in the calvaria, and Fgfs-<em>20</em> and -9 in the developing limbs, suggestive of their role in proliferation, differentiation and apoptosis.

We investigated whether adipose-derived adult stromal (ADAS) are of neural crest origin and the extent to which Notch 1 regulates their <em>growth</em> and differentiation. Mouse ADAS cells cultured in media formulated for neural stem cells (NSC) displayed limited capacity for self-renewal, clonogenicity, and neurosphere formation compared to NSC from the subventricular zone in the hippocampus. Although ADAS cells expressed Nestin, GFAP, NSE and Tuj1 in vitro, exposure to NSC differentiation supplements did not induce mature neuronal marker expression. In contrast, in mesenchymal stem cell (MSC) media, ADAS cells retained their ability to proliferate and differentiate beyond <em>20</em> passages and expressed high levels of Nestin. In neuritizing cocktails, ADAS cells extended processes, downregulated Nestin expression, and displayed depolarization-induced Ca(2+) transients but no spontaneous or evoked neural network activity on Multi-Electrode Arrays. Deletion of Notch 1 in ADAS cell cultures grown in NSC proliferation medium did not significantly alter their proliferative potential in vitro or the differentiation-induced downregulation of Nestin. Co-culture of ADAS cells with <em>fibroblasts</em> that stably expressed the Notch ligand Jagged 1 or overexpression of the Notch intracellular domain (NICD) did not alter ADAS cell <em>growth</em>, morphology, or cellular marker expression. ADAS cells did not display robust expression of neural crest transcription <em>factors</em> or genes (Sox, CRABP2, and TH); and lineage tracing analyses using Wnt1-Cre;Rosa26R-lacZ or -EYFP reporter mice confirmed that fewer than 2% of the ADAS cell population derived from a Wnt1-positive population during development. In summary, although media formulations optimized for MSCs or NSCs enable expansion of mouse ADAS cells in vitro, we find no evidence that these cells are of neural crest origin, that they can undergo robust terminal differentiation into functionally mature neurons, and that Notch 1 is likely to be a key regulator of their cellular and molecular characteristics.

It is unknown whether long-term pharmacological stimulation of soluble guanylate cyclase (sGC), elevating intracellular cGMP levels, has a beneficial effect on hypertension. The purpose of this study is to investigate the effects of BAY41-2272, an orally available sGC stimulator, on cardiovascular remodeling in hypertensive rats. Eight-week-old male Wistar rats with hypertension induced by angiotensin II infused subcutaneously at 250 ng/kg per minute were treated orally with a low ([L] 2 mg/kg per day) or high ([H] 10 mg/kg per day) dose of BAY41-2272 for 14 days. BAY41-2272-H partially suppressed the rise in blood pressure and reduced the heart weight (4.<em>20</em>+/-0.34 versus 3.68+/-0.<em>20</em> mg/g; P<0.01), whereas BAY41-2272-L had no effect. However, both doses decreased the angiotensin II-induced left ventricular accumulation of collagen in the perivascular area (L, -<em>20</em>%, P<0.05; H, -30%, P<0.01) and myocardial interstitium (L, -21%, P<0.05; H, -38%, P<0.01), reducing the number of activated <em>fibroblasts</em> surrounding coronary arteries (L, -74%; H, -79%; P<0.05). BAY41-2272 downregulated the angiotensin II-induced left ventricular gene expression of type 1 collagen (L, -41%, P<0.05; H, -49%, P<0.01) and transforming <em>growth</em> <em>factor</em>-beta1 (L, -49%, P<0.05; H, -65%, P<0.01). cGMP levels were elevated by BAY41-2272 not only in the left ventricle, but also in cultured cardiac <em>fibroblasts</em>, resulting in reduced thymidine incorporation into the cells. Thus, stimulation of sGC by BAY41-2272 attenuates fibrosis of the left ventricle in rats with angiotensin II-induced hypertension partly in a pressure-independent manner, suggesting an important role for sGC generating cGMP in inhibiting cardiovascular remodeling.

<em>Growth</em> <em>factors</em> induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. <em>Growth</em> <em>factors</em> initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble ( approximately <em>20</em> min). Considerable attention has been devoted to the mechanism by which <em>growth</em> <em>factors</em> induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G(12) and G(13) control the rate of disassembly of dorsal ruffles. In Galpha(12)(-/-)Galpha(13)(-/-) <em>fibroblast</em> cells, dorsal ruffles induced by <em>growth</em> <em>factor</em> treatment remain visible substantially longer ( approximately 60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without Galpha(12) and Galpha(13). Thus, Galpha(12)/Galpha(13) critically regulate dorsal ruffle turnover.

OBJECTIVE

Focal adhesion kinase (FAK) is involved in processes integral to angiogenesis, such as cell growth, survival, and migration. FAK is activated by angiogenic growth factors, such as insulin-like growth factor (IGF)-I, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The study was conducted to determine whether overexpression of FAK or FAK-related nonkinase (FRNK), an inhibitor of FAK, could influence human retinal endothelial cell (HREC) migration and in vivo angiogenesis.

METHODS

Migration in response to a combination of growth factors was examined in transfected HRECs overexpressing FAK or FRNK. The effect of FAK or FRNK overexpression on preretinal neovascularization was examined in a mouse model of oxygen-induced retinopathy.

RESULTS

Overexpression of FAK in HRECs resulted in a 102% +/- 13% increase (P = 1.4 x 10(-4)) in cell migration, whereas overexpression of FRNK resulted in a 20% +/- 8% decrease (P = 0.01). Overexpression of FAK in mouse eyes led to formation of numerous large vascular tufts resembling glomeruli and a 57% +/- 7% increase in preretinal neovascularization (P = 3 x 10(-9)), whereas FRNK resulted in a 55% +/- 15% reduction (P = 5 x 10(-5)).

CONCLUSIONS

Modulating the FAK/FRNK system may provide a novel approach to inhibiting pathologic retinal angiogenesis.

Accumulating evidence suggests that the delivery of human adipose tissue-derived stromal cells (hASCs) has great potential as regenerative therapy. This was performed to develop a method for expanding hASCs by reducing the amount of serum required. We demonstrate that hASCs were able to expand efficiently in media containing 2% serum and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2. These cells, or low serum cultured hASCs (hLASCs), expressed cell surface markers similar to those on bone marrow-derived mesenchymal stem cells, and could be differentiated into cells of mesenchymal lineage. Of interest, hLASCs secreted higher levels of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) than hASCs cultured in <em>20</em>% serum (hHASCs). Moreover, hLASC-conditioned media significantly increased endothelial cell (EC) proliferation and decreased EC apoptosis compared to that obtained from hHASCs or control media only. Antibodies against VEGF and HGF virtually negated these effects. When hASCs were administered into the ischemic hindlimbs of nude rats, hLASCs improved blood flow, increased capillary density, and raised the levels of VEGF and HGF in the muscles as compared with hHASCs. In conclusion, we demonstrate a novel low serum culture system for hASCs, which may have great potential in regenerative cell therapy for damaged organs in the clinical setting.

Vitamin D deficiency ((<em>20</em> ng/mL) and insufficiency (<em>20</em>-29 ng/mL) are common among patients with chronic kidney disease (CKD) or undergoing dialysis. In addition to nutritional and sunlight exposure deficits, <em>factors</em> that affect vitamin D deficiency include race, sex, age, obesity and impaired vitamin D synthesis and metabolism. Serum 1,25(OH)₂D levels also decrease progressively because of 25(OH)D deficiency, together with impaired availability of 25(OH)D by renal proximal tubular cells, high <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-23 and decreased functional renal tissue. As in the general population, this condition is associated with increased morbidity and poor outcomes. Together with the progressive decline of serum calcitriol, vitamin D deficiency leads to secondary hyperparathyroidism (SHPT) and its complications, tertiary hyperparathyroidism and hypercalcemia, which require surgical parathyroidectomy or calcimimetics. Kidney Disease Outcomes Quality Initiative (KDOQI) and Kidney Disease Improving Global Outcomes (KDIGO) experts have recognized that vitamin D insufficiency and deficiency should be avoided in CKD and dialysis patients by using supplementation to prevent SHPT. Many vitamin D supplementation regimens using either ergocalciferol or cholecalciferol daily, weekly or monthly have been reported. The benefit of native vitamin D supplementation remains debatable because observational studies suggest that vitamin D receptor activator (VDRA) use is associated with better outcomes and it is more efficient for decreasing the serum parathormone (PTH) levels. Vitamin D has pleiotropic effects on the immune, cardiovascular and neurological systems and on antineoplastic activity. Extra-renal organs possess the enzymatic capacity to convert 25(OH)D to 1,25(OH)₂D. Despite many unanswered questions, much data support vitamin D use in renal patients. This article emphasizes the role of native vitamin D replacement during all-phases of CKD together with VDRA when SHPT persists.

Endogenous electric fields are instructive during embryogenesis by acting to direct cell migration, and postnatally, they can promote axonal <em>growth</em> after injury (McCaig 1991, Al-Majed <em>20</em>00). However, the mechanisms for these changes are not well understood. Application of an appropriate electrical stimulus may increase the rate and success of nerve repair by directly promoting axonal <em>growth</em>. Previously, DC electrical stimulation at 50 mV/mm (1 mA, 8 h duration) was shown to promote neurite out<em>growth</em> and a more pronounced effect was observed if both peripheral glia (Schwann cells) and neurons were co-stimulated. If electrical stimulation is delivered to an injury site, both the neurons and all resident non-neuronal cells [e.g., Schwann cells, endothelial cells, <em>fibroblasts</em>] will be treated and this biophysical stimuli can influence axonal <em>growth</em> directly or indirectly via changes to the resident, non-neuronal cells. In this work, non-neuronal cells were electrically stimulated, and changes in morphology and neuro-supportive cells were evaluated. Schwann cell response (morphology and orientation) was examined after an 8 h stimulation over a range of DC fields (0-<em>20</em>0 mV/mm, DC 1 mA), and changes in orientation were observed. Electrically prestimulating Schwann cells (50 mV/mm) promoted 30% more neurite out<em>growth</em> relative to co-stimulating both Schwann cells with neurons, suggesting that electrical stimulation modifies Schwann cell phenotype. Conditioned medium from the electrically prestimulated Schwann cells promoted a <em>20</em>% increase in total neurite out<em>growth</em> and was sustained for 72 h poststimulation. An 11-fold increase in nerve <em>growth</em> <em>factor</em> but not brain-derived neurotrophic <em>factor</em> or glial-derived <em>growth</em> <em>factor</em> was found in the electrically prestimulated Schwann cell-conditioned medium. No significant changes in <em>fibroblast</em> or endothelial morphology and neuro-supportive behavior were observed poststimulation. Electrical stimulation is widely used in clinical settings; however, the rational application of this cue may directly impact and enhance neuro-supportive behavior, improving nerve repair.

Cardiac <em>fibroblasts</em> are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac <em>fibroblasts</em> remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial <em>fibroblasts</em> through modulation of potassium channels. Biophysical property of potassium channels in human atrial <em>fibroblasts</em> was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca(2+)-activated K(+) current (BK(Ca)), transient outward K(+) current (I(to)) and inwardly rectifying K(+) current (IK(ir)) were found in human atrial <em>fibroblasts</em>. Current density of BK(Ca) (IC50 = 69.4 μM; n = 6), I(to) (IC50 = 55.1 μM; n = 6) and IK(ir) (IC50 = 78.9 μM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial <em>fibroblasts</em>. Furthermore, NaHS (100-500 μM) inhibited <em>fibroblast</em> proliferation induced by transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1; 1 ng/ml), Ang II (100 nM) or <em>20</em>% FBS. Pre-conditioning of <em>fibroblasts</em> with NaHS decreased basal expression of Kv4.3 (encode I(to)), but not KCa1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)). Furthermore, H2S significantly attenuated TGF-β1-stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-β-induced myo<em>fibroblast</em> transformation. Our results show that H2S attenuates atrial <em>fibroblast</em> proliferation via suppression of K(+) channel activity and moderates their differentiation towards myo<em>fibroblasts</em>.

OBJECTIVE

The concept of neovascularization in response to tissue ischemia was recently extended by the finding of postnatal vasculogenesis through circulating endothelial progenitor cells (EPCs). The aim of this study was to assess the role of acute ischemia for EPC mobilization in patients with peripheral arterial occlusive disease (PAOD) and in healthy volunteers.

METHODS

The number of circulating EPCs was analyzed by flow cytometry in PAOD patients (n = 23) with exercise-induced limb ischemia for up to 72 h after a maximal treadmill test and in healthy volunteers (n = 17) who underwent a 15-min suprasystolic occlusion of one lower extremity to induce limb ischemia. Plasma concentrations of vascular endothelial growth factor, basic fibroblast growth factor, tumor necrosis factor-α, and granulocyte macrophage-colony stimulating factor were determined by ELISA.

RESULTS

EPCs (CD 34 pos/KDRpos) increased significantly in both PAOD patients from 82 ± 20 to 256 ± 52 (P < 0.05) and healthy volunteers from 144 ± 39 to 590 ± 61 cells per 1 million events (P < 0.05) in response to induced ischemia, with a maximum after 24 h and returned to baseline within 72 h. The relative increase in EPC numbers was significantly lower in patients with PAOD as compared with healthy volunteers (P < 0.05). Plasma levels of vascular endothelial growth factor increased from 27.4 ± 3.1 to 126.4 ± 12 pg/ml in patients with PAOD (P < 0.05) and from 30.7 ± 6.1 to 134.1 ± 12.4 pg/ml in healthy volunteers (P < 0.05).

CONCLUSIONS

Both patients with symptomatic PAOD and healthy volunteers respond to a single episode of limb ischemia with a time-dependent increase in circulating EPCs. The increase of EPC numbers in response to ischemia is reduced when vascular disease is present, underlining the reduced vasculogenic potential of patients with PAOD.

The prognosis of follicular lymphoma could vary with the tumor immune microenvironment. We evaluated the prognostic value of serum levels of ten cytokines. Our study cohort included 60 follicular lymphoma patients and <em>20</em> controls. Serum was available at diagnosis in 31 patients, at first relapse in 18, and complete remission in 11. Bioplex technology was used for determination of nine cytokines [interleukin (IL)-1Ra, IL-6, IL-7, IL-10, IL-13, tumor necrosis <em>factor</em> alpha (TNF-α), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF)]. Transforming <em>growth</em> <em>factor</em> beta (TGF-β) was measured by sandwich enzyme-linked immunosorbent assay. IL-1Ra, IL-6, IL-7, IL-10, IL-13, TNF-α, VEGF, and PDGF levels were found increased in follicular lymphoma patients compared to controls. Multivariate analysis identified early stage and high TGF-β levels as independent predictors of overall survival associated with improved outcome. High lactate dehydrogenase and VEGF levels were independently associated with poorer progression-free survival. These results show the prognostic value of TGF-β and VEGF in follicular lymphoma and suggest their contribution to tumor microenvironment alterations.

A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in (<em>20</em> min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>, and tetanus toxin C-fragment.

Fluorescent protein probes now permit spatial distributions of specific intracellular signaling molecules to be observed in real time. Mathematical models have been used to simulate molecular gradients and other spatial patterns within cells, and the output of such models may be compared directly with experiments if the binding of the fluorescent probe and the physics of the imaging technique are each incorporated. Here we present a comprehensive model describing the dynamics of 3' phosphoinositides (PIs), lipid second messengers produced in the plasma membrane in response to stimulation of the PI 3-kinase signaling pathway, as monitored in the cell-substratum contact area using total internal reflection fluorescence microscopy. With this technique it was previously shown that uniform stimulation of <em>fibroblasts</em> with platelet-derived <em>growth</em> <em>factor</em> elicits the formation of axisymmetric 3' PI gradients, which we now characterize in the context of our model. We find that upper and lower bounds on the relevant dimensionless model parameter values for an individual cell can be calculated from four well-defined fluorescence measurements. Based on our analysis, we expect that the key dimensionless group, the ratio of 3' PI turnover and diffusion rates, can be estimated within approximately <em>20</em>% or less.

Cellular and molecular mechanisms responsible for the observed vulnerary effects of recombinant human platelet-derived <em>growth</em> <em>factor</em> BB (rP-DGF-BB) in man have not been elucidated. In a double-blinded trial, patients having chronic pressure ulcers were treated topically with either rPDGF-BB or placebo for 28 days. To explore how rPDGF-BB may induce chronic wounds to heal, biopsies were taken from the ulcers of a cohort of <em>20</em> patients from the trial and evaluated in a blinded fashion by light microscopy for 1), <em>fibroblast</em> content, 2) neovessel formation, and 3), collagen deposition. Electron microscopy also was used to assess <em>fibroblast</em> activation and collagen deposition. Before initiation of therapy most wounds had few <em>fibroblasts</em> and most of those present were not activated. When mean scores for the total active treatment phase (days 8, 15, and 29) for rPDGF-BB-treated ulcers were compared with the scores for placebo-treated ulcers, <em>fibroblast</em> content was significantly higher for the rPDGF-BB-treated ulcers (P = 0.03, Kruskal-Wallis test). More significant differences in <em>fibroblast</em> and neovessel content were observed when six nonhealing wounds were eliminated from the analysis (three placebo, three treatment). Thus, in all healing wounds, rPDGF-BB therapy significantly increased <em>fibroblast</em> (P = 0.0007) and neovessel (P = 0.02) content. These results were correlated with increased collagen fibrillogenesis by <em>fibroblasts</em> from healing rPDGF-BB-treated wounds, as assessed by intracellular procollagen type I immunostaining, and by electron microscopy, and were concordant with clinical measurements (eg, area of ulcer opening and ulcer volume) which showed greater healing in rPDGF-BB-treated wounds. These results suggest induction of <em>fibroblast</em> proliferation and differentiation is one mechanism by which rPDGF-BB can accelerate wound healing and that rPDGF-BB can augment healing responses within a majority of, but not all, nonhealing chronic pressure ulcers in man.

Insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) stimulates the <em>growth</em> of thyroid cells of different animal species. However, conflicting results have been reported as regards the function and mechanism of the action of IGF-I at the thyroid level. This study was designed to determine whether normal human thyroid cells have IGF-I receptors and whether IGF-I could act on such cells through an autocrine mechanism. Human thyroid follicular cells in primary culture, not contaminated by <em>fibroblasts</em>, were used. They had specific and saturable binding sites for IGF-I, as revealed by radiolabeled binding method, and displayed an average receptor number of <em>20</em>00/cell. Under the same experimental conditions, insulin receptors were not detectable. Human thyroid follicular cells secreted IGF-I into the culture medium, as assessed by a specific chemiluminescent immunoassay. The IGF-I secretory process was detectable for at least 12 days of culture, but a high degree of variability has been found among individual samples. Acid-gel chromatography demonstrated that IGF-I and a higher mol wt IGF-I, most likely IGF-I bound to its binding protein, were secreted by human thyroid cells. TSH stimulated the secretion of the two molecules in normal human thyroid cells. The TSH effect on IGF-I secretion was concentration dependent between 0.1 nmol/L and 0.1 mumol/L. GH stimulated IGF-I synthesis by thyroid cells in a concentration range from <em>20</em>-<em>20</em>0 micrograms/mL. Since binding studies demonstrated the presence of IGF-I receptors on human thyroid cells, IGF-I probably regulates human thyroid function through an autocrine mechanism.

Hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) is considered to be a mesenchymal-derived <em>factor</em> that acts via a dual system receptor, consisting of the MET receptor and proteoglycans present on adjacent epithelial cells. Surprisingly, HGS/SF stimulated the migration of rat mammary (Rama) 27 <em>fibroblasts</em>, although it failed to stimulate their proliferation. HGF/SF stimulated a transient activation of mitogen-activated protein kinases p44 and p42 (p42/44(MAPK)), with a maximum level of dual phosphorylation of p42/44(MAPK) occurring 10-15 min after the addition of the <em>growth</em> <em>factor</em>, which was followed by a rapid decrease to near basal levels after <em>20</em> min. Interestingly, a second phase of p42/44(MAPK) dual phosphorylation was observed at later times (3 h to 10 h). PD098059, a specific inhibitor of MEK-1, prevented the dual phosphorylation of p42/44(MAPK) and also the phosphorylation of p90(RSK) (ribosomal subunit S6 kinase), which mirrored the kinetics of p42/44(MAPK) phosphorylation. Moreover, PD098059 prevented the HGF/SF-induced migration of Rama 27 cells. HGF/SF also induced an early increase in the phosphorylation of protein kinase B/Akt. Akt phosphorylation was elevated 15 min after the addition of HGF/SF and then declined to basal levels by 30 min. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), prevented the increase in Akt phosphorylation and abolished HGF/SF-induced migration of <em>fibroblasts</em>. PD098059 also inhibited the stimulation of Akt phosphorylation by HGF/SF and wortmannin similarly inhibited the stimulation of p42/44(MAPK) dual phosphorylation. These results suggest that HGF/SF-induced motility depends on both the transient dual phosphorylation of p42/44(MAPK) and the activation of PtdIns3K in Rama 27 <em>fibroblasts</em> and that these pathways are mutually dependent.

The Ets family of transcription <em>factors</em> are important downstream targets in cellular transformation, as altering Ets activity has been found to reverse the transformed phenotype of Ras transformed mouse <em>fibroblasts</em> and of several human tumor cell lines. To determine whether Ets <em>factors</em> are important targets in the largely uncharacterized aberrant signaling in prostate cancer, we have altered Ets activity in the prostate tumor cell line PPC-1, by stable expression of either full-length Ets2, or a dominant inhibitor of Ets activity, the Ets2 DNA binding domain (Ets2DBD). Analysis of multiple independent clonal cell lines revealed that expression of either Ets2 or the Ets2DBD inhibited the anchorage-independent <em>growth</em> of PPC-1 cells up to <em>20</em>-fold. In contrast to our previous findings with Ras-transformed NIH3T3 cells, PPC-1 cell lines expressing either Ets2 or the EtsDBD exhibited slower attached cell <em>growth</em>, increased Ets-dependent gene expression, and up to a 10-fold increase in apoptotic cell death. The p21cip gene was identified as a potential target of altered Ets signaling. Interestingly, the two distinct Ets2 constructs had strikingly different effects on in vitro invasiveness. Expression of the Ets2DBD almost completely blocked PPC-1 cell invasion through Matrigel, whereas over-expression of full-length Ets2 did not inhibit invasion. Overall, these results demonstrate that the balance of Ets <em>factor</em> activity can regulate multiple aspects of the transformed phenotype of PPC-1 prostate tumor cells, including anchorage-independent <em>growth</em>, survival, and invasiveness.

OBJECTIVE

To determine the mechanism by which the bile acid sequestrant colesevelam improves glycemic control.

METHODS

We performed a frequently sampled intravenous glucose tolerance test (FSIGT) with minimal model analysis and a meal tolerance test (MTT) in <em>20</em> subjects with impaired fasting glucose (11 men, 9 women; mean age 60.7 ± 1.9 years, BMI 29.4 ± 0.9 kg/m(2)) in a single-blind study after 2 weeks of placebo treatment and 8 weeks of colesevelam 3.75 g daily. From these tests, insulin sensitivity, β-cell function, and glucose tolerance were determined, along with gastrointestinal peptide levels during the MTT.

RESULTS

Fasting plasma glucose and HbA(1c) decreased with colesevelam (from 5.9 ± 0.1 to 5.7 ± 0.1 mmol/L, P < 0.05, and from 5.86 ± 0.06 to 5.76 ± 0.06%, P = 0.01, respectively), but fasting insulin did not change. Colesevelam had no effect on any FSIGT measures. In contrast, the MTT incremental area under the curve (iAUC) for both glucose (from 249.3 ± 28.5 to 198.8 ± 23.6 mmol/L · min, P < 0.01) and insulin (from <em>20</em>,130 [13,542-35,292] to 13,086 [9,804-21,138] pmol/L · min, P < 0.05) decreased with colesevelam. However, the ratio of iAUC insulin to iAUC glucose was not changed. iAUC for cholecystokinin (CCK) increased (from 43.2 [0-130.1] to 127.1 [47.2-295.2] pmol/L · min, P < 0.01), while iAUC for fibroblast growth factor 19 decreased (from 11,185 [1,346-17,661] to 2,093 [673-6,707] pg/mL · min, P < 0.01) with colesevelam. However, iAUC for glucagon, glucose-dependent insulinotropic peptide, and glucagon-like peptide 1 did not change.

CONCLUSIONS

Colesevelam improves oral but not intravenous glucose tolerance without changing insulin sensitivity, β-cell function, or incretins. This effect may be at least partially explained by the colesevelam-induced increase in CCK.

BACKGROUND

Tumour microvascularity is a significant determinant of prognosis for a large number of different tumours, including uveal melanoma. The development of blood vessels within these and other tumours is partly controlled by soluble pro-angiogenic cytokines, of which basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF) are the best described.

METHODS

Because VEGF has been inconsistently found within uveal melanomas and bFGF is described as an autocrine growth factor in cutaneous melanoma, the authors looked at the expression of these cytokines in uveal melanomas using immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The cross talk between uveal melanoma cells and endothelial cells was then assessed in an in vitro co-culture model.

RESULTS

While most tumour cells expressed bFGF at the protein level by immunohistochemistry (89%), relatively few (22%) expressed VEGF, and this was of limited extent. All 20 tumours tested by RT-PCR contained mRNA for both bFGF and VEGF. Co-culture experiments using an ATP based bioassay showed that uveal melanomas could support the growth of a rat brain endothelial cell line (GPNT) and human umbilical vein endothelial cells (HUVEC), and that this could be modulated by cytokines and anti-cytokine antibodies.

CONCLUSIONS

These results suggest that angiogenesis within uveal melanoma may be the result of a complex interplay between endothelial and tumour cells, and that bFGF and VEGF could play a part.

Secreted soluble <em>growth</em> <em>factors</em> have long been implicated in regulating or modulating cortical neurogenesis through stimulation of neurogenic progenitors. How a cortical progenitor cell interprets the <em>growth</em> <em>factor</em> signal may determine the progeny produced by the progenitor through a critical final round of cell division prior to terminal differentiation. Given that low concentrations of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) have previously been shown to stimulate cortically derived rodent progenitors to produce neuroblasts, we hypothesized that low levels of FGF-2 may also promote neurogenesis from human neural progenitor cells (hNPC). hNPC were generated from the developing human cortex and maintained in epidermal <em>growth</em> <em>factor</em> as neurospheres. CREB phosphorylation revealed that a similar number of differentiating hNPC were stimulated by both low (2 pg/ml) and high (<em>20</em> ng/ml) levels of FGF-2. The majority of progenitor cells that produced neurons underwent a final round of division during differentiation. Low concentrations of FGF-2 increased neurogenesis while high levels of FGF-2 maintained progenitor cell proliferation and blocked neurogenesis. Application of an FGF-2 neutralizing antibody during differentiation completely inhibited CREB phosphorylation but did not block neurogenesis. Thus, while low levels of FGF-2 can increase neurogenesis; extracellular stimulation by this <em>factor</em> is not required for new neuron production from hNPC.

Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1 beta (IL1 beta) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1 beta, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from <em>20</em>-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and <em>20</em>-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and <em>20</em>-day-old rats with FSH, IL1 beta, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones.

BACKGROUND

Previous studies have demonstrated that biological aging has a negative influence on the therapeutic effects of mesenchymal stem cells (MSCs)-based therapy. Using a rat myocardial infarction (MI) model, we tested the hypothesis that silent mating type information regulation 2 homolog 1 (SIRT1) may ameliorate the phenotype and improve the function of aged MSCs and thus enhance the efficacy of aged MSCs-based therapy.

METHODS

Sixty female rats underwent left anterior descending coronary artery ligation and were randomly assigned to receiving: intramyocardial injection of cell culture medium (DMEM group); SIRT1 overexpression vector-treated aged MSCs (SIRT1-aged MSCs group) obtained from aged male SD rats or empty vector-treated aged MSCs (vector-aged MSCs group). Another <em>20</em> sham-operated rats that underwent open-chest surgery without coronary ligation or any other intervention served as controls.

RESULTS

SIRT1-aged MSC group exhibited enhanced blood vessel density in the border zone of MI hearts, which was associated with reduced cardiac remodeling, leading to improved cardiac performance. Consistent with the in vivo data, our in vitro experiments also demonstrated that SIRT1 overexpression ameliorated aged MSCs senescent phenotype and recapitulated the pro-angiogenesis property of MSCs and conferred the anti-stress response capabilities, as indicated by increases in pro-angiogenic factors, angiopoietin 1 (Ang1) and basic fibroblast growth factor (bFGF), expressions and a decrease in anti-angiogenic factor thrombospondin-1 (TBS1) at mRNA levels, and increases in Bcl-2/Bax ratio at protein level.

CONCLUSIONS

Up-regulating SIRT1 expression could enhance the efficacy of aged MSCs-based therapy for MI as it relates to the amelioration of senescent phenotype and hence improved biological function of aged MSCs.