BACKGROUND

Vascular endothelial growth factor (VEGF), an endothelial cell mitogen that promotes angiogenesis, was initially identified as a vascular permeability factor (VPF). Abundant evidence suggests that angiogenesis is preceded and/or accompanied by enhanced microvascular permeability. The mechanism by which VEGF/VPF increases vascular permeability (VP), however, has remained enigmatic. Accordingly, we used an in vivo assay of VP (Miles assay) to study the putative mediators of VEGF/VPF-induced permeability.

RESULTS

VEGF/VPF and positive controls (platelet-activating factor [PAF], histamine, and bradykinin) all increased vascular permeability. Prior administration of the tyrosine kinase inhibitors genistein or herbimycin A prevented VEGF/VPF-induced permeability. Placenta growth factor, which binds to Flt-1/VEGF-R1 but not Flk-1/KDR/VEGF-R2 receptor tyrosine kinase, failed to increase permeability. Other growth factors such as basic fibroblast growth factor (FGF), acidic FGF, platelet-derived growth factor-BB, transforming growth factor-beta, scatter factor, and granulocyte macrophage-colony stimulating factor (8 to 128 ng) failed to increase permeability. VEGF/VPF-induced permeability was significantly attenuated by the nitric oxide (NO) synthase inhibitors N(omega)-nitro-L-arginine (10 mg/kg) or N(omega)-nitro-L-arginine methyl ester (20 mg/kg) and the cyclooxygenase inhibitor indomethacin (5 mg/kg). The inactive enantiomer N(omega)-nitro-D-arginine methyl ester (20 mg/kg) did not inhibit VEGF/VPF-induced permeability. In vitro studies confirmed that VEGF/VPF stimulates synthesis of NO and prostaglandin metabolites in microvascular endothelial cells. Finally, NO donors and the prostacyclin analogue taprostene administered together but not alone reproduced the increase in permeability observed with VEGF/VPF.

CONCLUSIONS

These results implicate NO and prostacyclin produced by the interaction of VEGF/VPF with its Flk-1/KDR/VEGF-R2 receptor as mediators of VEGF/VPF-induced vascular permeability. Moreover, this property appears unique to VEGF/VPF among angiogenic cytokines.

OBJECTIVE

To investigate whether adult human articular chondrocytes (AHACs), dedifferentiated by monolayer expansion, can differentiate toward diverse mesenchymal lineages and, if so, whether this ability is regulated by growth factors during monolayer expansion.

METHODS

AHACs were expanded as multiclonal or clonal populations in medium without (control) or with factors enhancing cell dedifferentiation (transforming growth factor beta1, fibroblast growth factor 2, and platelet-derived growth factor type BB [TFP]). Cells were then cultured under conditions promoting chondrogenic, osteogenic, or adipogenic differentiation, and the acquired phenotypes were assessed histologically, biochemically, and by real-time reverse transcriptase-polymerase chain reaction.

RESULTS

Multiclonal populations of both control- and TFP-expanded AHACs differentiated toward the chondrogenic, osteogenic, and adipogenic lineages. Compared with control-expanded AHACs, TFP-expanded cells displayed enhanced chondrogenic differentiation capacity (2.4-fold higher glycosaminoglycan/DNA content and 2,500-fold higher up-regulation of type II collagen) and osteogenic differentiation capacity (9.4-fold higher increase in alkaline phosphatase activity and 12.4-fold higher up-regulation of bone sialoprotein), but reduced formation of adipocytes (5.2-fold lower oil red O-positive cells/area). Clonal populations of AHACs could be efficiently expanded in TFP, but not in control medium. Most TFP-expanded clones were able to redifferentiate only into chondrocytes (7 of 20) or were unable to differentiate (6 of 20). However, some clones (2 of 20) differentiated toward all of the lineages investigated, thus displaying characteristics of mesenchymal progenitor cells.

CONCLUSIONS

Dedifferentiated AHACs exhibit differentiation plasticity, which is modulated by growth factors used during monolayer expansion and is highly heterogeneous across different clones. Clonal culture of AHACs in the presence of regulatory molecules could lead to the identification of AHAC subpopulations with enhanced cartilage repair capacity.

BACKGROUND

Recent studies have suggested that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) may have synergistic effects on the induction of angiogenesis in vitro. Therefore, we investigated the hypothesis that the simultaneous administration of VEGF and bFGF, each having been previously shown to independently enhance collateral development in an animal model of hind limb ischemia, could have a synergistic effect in vivo.

RESULTS

Ten days after surgical induction of unilateral hind limb ischemia, New Zealand White rabbits were randomized to receive either VEGF 500 micrograms alone (n = 6), bFGF 10 micrograms alone (n = 7), VEGF 500 micrograms, immediately followed by 10 micrograms bFGF (n = 7), or vehicle only (control animals, n = 8) in each case administered intra-arterially via a catheter in the internal iliac artery of the ischemic limb. BP ratio (BPR, ischemic/healthy limb) at day 10 for the VEGF+bFGF group was 0.82 +/- 0.01, much superior (P < .0005) to that of either the VEGF group (0.52 +/- 0.02) or the bFGF group (0.57 +/- 0.02). This outcome persisted at day 30: BPR in the VEGF+bFGF group (0.91 +/- 0.02) exceeded that of the control group (0.49 +/- 0.05, P < .0001), the VEGF group (0.65 +/- 0.03, P < .0005), or the bFGF group (0.66 +/- 0.03, P < .0005). Serial angiography demonstrated a progressive increase in luminal diameter of the stem collateral artery and the number of opacified collaterals in the thigh of the ischemic limbs in all groups. Stem artery diameter with VEGF+bFGF (1.34 +/- 0.07 mm) on day 30 was significantly (P < .05) greater than with either VEGF (1.09 +/- 0.09) or bFGF (1.18 +/- 0.06) alone. Capillary density was significantly greater (P < .05) in VEGF+bFGF animals (275 +/- 20 mm2) compared with VEGF (201 +/- 8) or bFGF (209 +/- 15).

CONCLUSIONS

Combined administration of VEGF and bFGF stimulates significantly greater and more rapid augmentation of collateral circulation, resulting in superior hemodynamic improvement compared with either VEGF or bFGF alone. This synergism of two angiogenic mitogens with different target cell specificities may have important implications for the treatment of severe arterial insufficiency in patients whose disease is not amenable to direct revascularization.

BACKGROUND

The present article is a report of our animal experiments and also of the first clinical results of a new treatment for coronary heart disease using the human growth factor FGF-I (basic fibroblast growth factor) to induce neoangiogenesis in the ischemic myocardium.

RESULTS

FGF-I was obtained from strains of Escherichia coli by genetic engineering, then isolated and highly purified. Several series of animal experiments demonstrated the apathogenic action and neoangiogenic potency of this factor. After successful conclusion of the animal experiments, it was used clinically for the first time. FGF-I (0.01 mg/kg body weight) was injected close to the vessels after the completion of internal mammary artery (IMA)/left anterior descending coronary artery (LAD) anastomosis in 20 patients with three-vessel coronary disease. All the patients had additional peripheral stenoses of the LAD or one of its diagonal branches. Twelve weeks later, the IMA bypasses were selectively imaged by intra-arterial digital subtraction angiography and quantitatively evaluated. In all the animal experiments, the development of new vessels in the ischemic myocardium could be demonstrated angiographically. The formation of capillaries could also be demonstrated in humans and was found in all cases around the site of injection. A capillary network sprouting from the proximal part of the coronary artery could be shown to have bypassed the stenoses and rejoined the distal parts of the vessel.

CONCLUSIONS

We believe that the use of FGF-I for myocardial revascularization is in principle a new concept and that it may be particularly suitable for patients with additional peripheral stenoses that cannot be revascularized surgically.

OBJECTIVE

To evaluate synovial fluid (SF) for the presence of mesenchymal progenitor cells (MPCs), to compare SF MPCs with bone marrow (BM) MPCs, and to enumerate these cells in both inflammatory arthritis and osteoarthritis (OA).

METHODS

SF from 100 patients with arthritis (53 rheumatoid arthritis [RA], <em>20</em> OA, and 27 other arthropathies) was evaluated. To establish multipotentiality, polyclonal and single cell-derived cultures of SF <em>fibroblasts</em> were examined by standard and quantitative differentiation assays. Their phenotype before and after expansion was determined by multiparameter flow cytometry. A colony-forming unit-<em>fibroblast</em> assay was used for SF MPC enumeration.

RESULTS

Regardless of the nature of the arthritis, both polyclonal and single cell-derived cultures of SF fibroblasts possessed trilineage mesenchymal differentiation potentials. The number of MPCs in a milliliter of SF was higher in OA (median 37) than in RA (median 2) (P < 0.00001). No significant differences in MPC numbers were found between early and established RA (median 3 and 2 cells/ml, respectively). Culture-expanded SF and BM MPCs had the same phenotype (negative for CD45 and positive for D7-FIB, CD13, CD105, CD55, and CD10). Rare, uncultured SF fibroblasts were CD45(low) and expressed low-affinity nerve growth factor receptor, similar to in vivo BM MPCs.

CONCLUSIONS

Our findings prove the presence of rare tripotential MPCs, at the single-cell level, in the SF of patients with arthritis. SF MPCs are clonogenic and multipotential fibroblasts that, despite the pathologic environment within a diseased joint, have a phenotype similar to that of uncultured BM MPCs. The higher prevalence of MPCs in OA SF suggests their likely origin from disrupted joint structures. These findings could determine the role of MPCs in the pathogenesis of inflammatory arthritis, together with their role in attempted joint regeneration in degenerative arthritis, which has yet to be established.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite which disrupts microtubule function, has been shown to inhibit proliferating cells in vitro and suppress certain murine tumors in vivo. In vitro screening has determined that breast cancer cell lines are most sensitive to inhibition by 2-ME. Additionally, 2-ME has been shown to inhibit angiogenesis in vitro. We tested whether 2-ME suppresses cytokine-induced angiogenesis in vivo and inhibits <em>growth</em> of a human breast carcinoma in severe combined immunodeficient mice. A model of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-induced corneal neovascularization in C57BL/6 mice was used to evaluate the antiangiogenic effects of 2-ME and other microtubule inhibitors such as Taxol, vincristine, and colchicine. 2-ME (150 mg/kg p.o., n = <em>20</em>) inhibited bFGF and VEGF-induced neovascularization by 39% and 54%, respectively. Taxol (6 mg/kg i.p., n = 17) inhibited bFGF and VEGF-induced neovascularization by 45% and 37%, respectively. Vincristine (0.2 mg/kg i.p., n = 8) and colchicine (0.25 mg/kg i.p., n = 8) had no effect. Treatment with 2-ME (75 mg/kg p.o., n = 9) for 1 month suppressed the <em>growth</em> of a human breast carcinoma in mice by 60% without toxicity. Recognition of the antiangiogenic and antitumor properties of 2-ME and Taxol may be crucial in planning clinical applications to angiogenesis-dependent diseases.

Injury to the central nervous system (CNS) by ionizing radiation may be a consequence of damage to the vascular endothelium. Recent studies showed that radiation-induced apoptosis of endothelial cells in vitro and in the lung in vivo is mediated by the lipid second messenger ceramide via activation of acid sphingomyelinase (ASM). This apoptotic response to radiation can be inhibited by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> or by genetic mutation of ASM. In the CNS, single-dose radiation has been shown to result in a 15% loss of endothelial cells within 24 h, but whether or not this loss is associated with apoptosis remains unknown. In the present studies, dose- and time-dependent induction of apoptosis was observed in the C57BL/6 mouse CNS. Apoptosis was quantified by terminal deoxynucleotidyl transferase-mediated nick end labeling, and specific endothelial apoptosis was determined by histochemical double labeling with terminal deoxynucleotidyl transferase-mediated nick end labeling and Lycopersicon esculentum lectin. Beginning at 4 h after single-dose radiation, apoptosis was ongoing for 24 h and peaked at 12 h at an incidence of 0.7-1.4% of the total cells in spinal cord sections. Up to <em>20</em>% of the apoptotic cells were endothelial. This effect was also seen in multiple regions of the brain (medulla, pons, and hippocampus). A significant reduction of radiation-induced apoptosis was observed after i.v. basic <em>fibroblast</em> <em>growth</em> <em>factor</em> treatment (0.45-4.5 microg/mouse). Identical results were noted in C3H/HeJ mice. Furthermore, irradiated ASM knockout mice displayed as much as a 70% reduction in endothelial apoptosis. This study demonstrates that ionizing radiation induces early endothelial cell apoptosis throughout the CNS. These data are consistent with recent evidence linking radiation-induced stress with ceramide and suggest approaches to modify the apoptotic response in control of radiation toxicity in the CNS.

<AbstractText><em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 2 gene alterations are involved in the pathogenesis of cholangiocarcinoma. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. This study evaluated the safety and antitumour activity of pemigatinib in patients with previously treated, locally advanced or metastatic cholangiocarcinoma with and without FGFR2 fusions or rearrangements.</AbstractText><AbstractText>In this multicentre, open-label, single-arm, multicohort, phase 2 study (FIGHT-<em>20</em>2), patients aged 18 years or older with disease progression following at least one previous treatment and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 recruited from 146 academic or community-based sites in the USA, Europe, the Middle East, and Asia were assigned to one of three cohorts: patients with FGFR2 fusions or rearrangements, patients with other FGF/FGFR alterations, or patients with no FGF/FGFR alterations. All enrolled patients received a starting dose of 13·5 mg oral pemigatinib once daily (21-day cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, withdrawal of consent, or physician decision. The primary endpoint was the proportion of patients who achieved an objective response among those with FGFR2 fusions or rearrangements, assessed centrally in all patients who received at least one dose of pemigatinib. This study is registered with ClinicalTrials.gov, NCT02924376, and enrolment is completed.</AbstractText><AbstractText>Between Jan 17, <em>20</em>17, and March 22, <em>20</em>19, 146 patients were enrolled: 107 with FGFR2 fusions or rearrangements, <em>20</em> with other FGF/FGFR alterations, 18 with no FGF/FGFR alterations, and one with an undetermined FGF/FGFR alteration. The median follow-up was 17·8 months (IQR 11·6-21·3). 38 (35·5% [95% CI 26·5-45·4]) patients with FGFR2 fusions or rearrangements achieved an objective response (three complete responses and 35 partial responses). Overall, hyperphosphataemia was the most common all-grade adverse event irrespective of cause (88 [60%] of 146 patients). 93 (64%) patients had a grade 3 or worse adverse event (irrespective of cause); the most frequent were hypophosphataemia (18 [12%]), arthralgia (nine [6%]), stomatitis (eight [5%]), hyponatraemia (eight [5%]), abdominal pain (seven [5%]), and fatigue (seven [5%]). 65 (45%) patients had serious adverse events; the most frequent were abdominal pain (seven [5%]), pyrexia (seven [5%]), cholangitis (five [3%]), and pleural effusion (five [3%]). Overall, 71 (49%) patients died during the study, most frequently because of disease progression (61 [42%]); no deaths were deemed to be treatment related.</AbstractText><AbstractText>These data support the therapeutic potential of pemigatinib in previously treated patients with cholangiocarcinoma who have FGFR2 fusions or rearrangements.</AbstractText><AbstractText>Incyte Corporation.</AbstractText>

Recent data suggest that uric acid is generated locally in the vessel wall by the action of xanthine oxidase. This enzyme, activated during ischemia/reperfusion by proteolytic conversion of xanthine dehydrogenase, catalyzes the oxidation of xanthine, thereby generating free radicals and uric acid. Because of the potential role of ischemia/reperfusion in vascular disease, we studied the effects of uric acid on rat aortic vascular smooth muscle cell (VSMC) <em>growth</em>. Uric acid stimulated VSMC DNA synthesis, as measured by [3H]thymidine incorporation, in a concentration-dependent manner with half-maximal activity at 150 microM. Maximal induction of DNA synthesis by uric acid (250 microM) was approximately 70% of 10% calf serum and equal to 10 ng/ml platelet-derived <em>growth</em> <em>factor</em> (PDGF) AB or <em>20</em> ng/ml <em>fibroblast</em> <em>growth</em> <em>factor</em>. Neither uric acid precursors (xanthine and hypoxanthine) nor antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) were mitogenic for VSMC. Uric acid was mitogenic for VSMC but not for <em>fibroblasts</em> or renal epithelial cells. The time course for uric acid stimulation of VSMC <em>growth</em> was slower than serum, suggesting induction of an autocrine <em>growth</em> mechanism. Exposure of quiescent VSMC to uric acid stimulated accumulation of PDGF A-chain mRNA (greater than 5-fold at 8 h) and secretion of PDGF-like material in conditioned medium (greater than 10-fold at 24 h). Uric acid-induced [3H]thymidine incorporation was markedly inhibited by incubation with anti-PDGF A-chain polyclonal antibodies. Thus uric acid stimulates VSMC <em>growth</em> via an autocrine mechanism involving PDGF A-chain. These findings suggest that generation of uric acid during ischemia/reperfusion contributes to atherogenesis and intimal proliferation following arterial injury.

Overexpression and activation of receptor tyrosine kinases, such as platelet derived <em>growth</em> <em>factor</em> receptors (PDGFRs) and epidermal <em>growth</em> <em>factor</em> receptor (EGFR), leads to proliferation of human malignant astrocytoma cells. Although oncogenic mutations affecting Ras are not prevalent in human malignant astrocytomas, we have investigated whether levels of activated Ras.GTP might be elevated in these tumors secondary to the mitogenic signals originating from activated receptor tyrosine kinases. In support of this hypothesis high levels of Ras.GTP, similar to those found in oncogenic Ras transformed <em>fibroblasts</em>, were present in four established human malignant astrocytoma cell lines which express PDGFRs and EGFR, and <em>20</em> operative malignant astrocytoma specimens. Stimulation of PDGFR's and EGFR's induced tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2, suggesting a mechanism by which Ras may be activated in human malignant astrocytoma cells. Furthermore, blocking Ras activation by expression of the Ha-Ras-Asn17 dominant-negative mutant, or by farnesyl transferase inhibitors, decreased in vitro proliferation of the human astrocytoma cell lines. These results support the hypothesis that proliferative signals from receptor tyrosine kinases expressed by human malignant astrocytoma cells utilize the Ras mitogenic pathway. Pharmacological inhibitors of the Ras pathway may therefore be of therapeutic value in these presently terminal tumors.

OBJECTIVE

It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype.

METHODS

CD34-/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity >90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum).

RESULTS

After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel the formation of cord- and tubular-like structures was observed.

CONCLUSIONS

The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.

An increased circulating level of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) is an independent risk <em>factor</em> for mortality, cardiovascular disease, and progression of chronic kidney disease (CKD), but its role in transplant allograft and patient survival is unknown. We tested the hypothesis that increased FGF23 is an independent risk <em>factor</em> for all-cause mortality and allograft loss in a prospective cohort of 984 stable kidney transplant recipients. At enrollment, estimated GFR (eGFR) was 51 ± 21 ml/min per 1.73 m(2) and median C-terminal FGF23 was 28 RU/ml (interquartile range, <em>20</em> to 43 RU/ml). Higher FGF23 levels independently associated with increased risk of the composite outcome of all-cause mortality and allograft loss (full model hazard ratio: 1.46 per SD increase in logFGF23, 95% confidence interval: 1.28 to 1.68, P<0.001). The results were similar for each component of the composite outcome and in all sensitivity analyses, including prespecified analyses of patients with baseline eGFR of 30 to 90 ml/min per 1.73 m(2). In contrast, other measures of phosphorus metabolism, including serum phosphate and parathyroid hormone (PTH) levels, did not consistently associate with outcomes. We conclude that a high (or elevated) FGF23 is an independent risk <em>factor</em> for death and allograft loss in kidney transplant recipients.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is a major mitogen for endothelial cells and enhances vascular permeability. Enhanced VEGF secretion is found in human cancers and correlates with increased tumor neovascularization. ZD6474 is a p.o. bioavailable, VEGF flk-1/KDR receptor (VEGFR-2) tyrosine kinase inhibitor with antitumor activity in many human cancer xenografts and is currently in Phase I clinical development.

METHODS

We tested the effects of ZD6474 on EGFR phosphorylation in cell expressing functional epidermal growth factor receptor (EGFR) and the antiproliferative and the proapoptotic activity of ZD6474 alone or in combination taxanes in human cancer cell lines with functional EGFR but lacking VEGFR-2. The antitumor activity of this drug was also tested in nude mice bearing established GEO colon cancer xenografts.

RESULTS

ZD6474 causes a dose-dependent inhibition of EGFR phosphorylation in mouse NIH-EGFR fibroblasts and human MCF-10A ras breast cancer cells, two cell lines that overexpress the human EGFR. ZD6474 treatment resulted in a dose-dependent inhibition of soft agar growth in seven human cell lines (breast, colon, gastric, and ovarian) with functional EGFR but lacking VEGFR-2. A dose-dependent supra-additive effect in growth inhibition and in apoptosis in vitro was observed by the combined treatment with ZD6474 and paclitaxel or docetaxel. ZD6474 treatment of nude mice bearing palpable GEO colon cancer xenografts (which are sensitive to inhibition of EGFR signaling) induced dose-dependent tumor growth inhibition. Immunohistochemical analysis revealed a significant dose-dependent reduction of neoangiogenesis. The antitumor activity of ZD6474 in GEO tumor xenografts was also found to be enhanced when combined with paclitaxel. Tumor regression was observed in all mice after treatment with ZD6474 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of angiogenesis. Six of 20 mice had no histological evidence of tumors after treatment with ZD6474 plus paclitaxel.

CONCLUSIONS

This study suggests that in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling, ZD6474 may also be able to inhibit cancer cell growth by blocking EGFR autocrine signaling. These results provide also a rationale for the clinical evaluation of ZD6474 combined with taxanes in cancer patients.

We investigated effects of the paracrine <em>factors</em> secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine <em>factors</em> in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-<em>20</em> times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic <em>factors</em> secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.

[<em>20</em>-(3)H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo <em>fibroblasts</em> in a specific, saturable, reversible fashion. Equilibrium binding occurred with a K(d) of 25 nM; this value is very close to the 50% effective dose (ED(50)), 50 nM, previously determined for the biological response (induction of fibronectin loss) in <em>growing</em> chicken embryo <em>fibroblasts</em>. At saturation, 1.4 pmol of [<em>20</em>-(3)H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 x 10(4) molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (K(i) = 2 nM), mezerein (K(i) = 180 nM), phorbol 12,13-dibenzoate (K(i) = 180 nM), phorbol 12,13-diacetate (K(i) = 1.7 muM), phorbol 12,13,<em>20</em>-triacetate (K(i) = 39 muM), and phorbol 13-acetate (K(i) = 1<em>20</em> muM). The measured K(i) values are all within a <em>factor</em> of 3.5 of the ED(50) values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 mug/ml) and 4alpha-phorbol 12,13-didecanoate (10 mug/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate <em>20</em>-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo <em>fibroblasts</em> are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 muM), phenol (1 mM), iodoacetic acid (1.7 muM), and cantharidin (75 muM), or by epidermal <em>growth</em> <em>factor</em> (100 ng/ml), dexamethasone acetate (2 muM), retinoic acid (10 muM), or prostaglandin E(2) (1 muM). These agents thus appear to act at a target distinct from that of the phorbol esters.

Vascular smooth muscle cells (VSMCs) proliferate in response to arterial injury. Recent findings suggest that, in addition to platelet-derived <em>growth</em> <em>factors</em>, <em>growth</em> <em>factors</em> from inflammatory cells and endothelial cells at the site of injury may contribute to VSMC proliferation. We hypothesized that a common mechanism by which endothelial cells and inflammatory cells stimulate VSMC <em>growth</em> could be the active oxygen species (i.e., O2-, H2O2, and .OH) generated during arterial injury. Using xanthine/xanthine oxidase to generate active oxygen species, we studied the effects of these agents on VSMC <em>growth</em>. Xanthine/xanthine oxidase (100 microM xanthine and 5 microunits/ml xanthine oxidase) stimulated DNA synthesis in <em>growth</em>-arrested VSMCs by 180% over untreated cells. Administration of the scavenging enzymes superoxide dismutase and catalase demonstrated that H2O2 was primarily responsible for xanthine/xanthine oxidase-induced VSMC DNA synthesis. H2O2 directly increased VSMC DNA synthesis and cell number (maximal at <em>20</em>0 microM) but decreased DNA synthesis of endothelial cells and <em>fibroblasts</em>. This effect was protein kinase C independent: sphingosine, a potent protein kinase C inhibitor, failed to block H2O2-induced VSMC DNA synthesis. H2O2 (<em>20</em>0 microM) stimulated c-myc and c-fos mRNA levels by fourfold and <em>20</em>-fold, respectively, as compared with quiescent levels. In contrast to DNA synthesis, H2O2 induction of c-myc and c-fos mRNA was primarily protein kinase C dependent. These findings show that H2O2 specifically increases VSMC DNA synthesis and suggest a role for this oxidant in intimal proliferation, especially after arterial injury.

An endothelial cell <em>growth</em> <em>factor</em> with unique specificity for vascular endothelial cells has been purified from the conditioned medium of the AtT-<em>20</em> pituitary cell line. This <em>growth</em> <em>factor</em>, which has been characterized as a homodimer composed of two subunits with mol. wts of 23 kd is a potent mitogen for vascular endothelial cells in vitro with activity detectable at 50 pg/ml and saturation at 1 ng/ml. It was also angiogenic in vivo. In contrast with other endothelial mitogens of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, it has a unique target cell specificity. It did not stimulate the <em>growth</em> of other cell types of the vascular system such as vascular smooth muscle cells or that of mesoderm and neuroectoderm derived cells. Microsequencing revealed an amino-terminal sequence with no homology to any known protein. The release of this novel endothelial cell <em>growth</em> <em>factor</em> by pituitary derived cells and its unique target cell specificity suggest that it could play an important role in the angiogenic process.

The effect of human epidermal <em>growth</em> <em>factor</em> (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the <em>growth</em> of human foreskin <em>fibroblasts</em> (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells <em>growing</em> in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the <em>growth</em> of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to <em>20</em>-fold increase in the amount of 3H-thymidine incorporation after <em>20</em>-24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of <em>growth</em>, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this <em>growth</em> <em>factor</em> on HF cells.

BACKGROUND

Hypoxia and indirect angiogenic factors may stimulate angiogenesis via induction of endothelial cell mitogen(s). To evaluate this hypothesis, we investigated whether low oxygen tension or cytokines known to promote neovascularization in vivo could modulate the expression of either vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) in human vascular smooth muscle cells (SMCs).

RESULTS

SMCs were treated with platelet-derived growth factor BB (PDGF-BB) or transforming growth factor-beta 1 (TGF-beta 1) or exposed to low oxygen tension in serum-free medium. Northern analysis detected low basal levels of VEGF and bFGF mRNA in extracts of unstimulated SMCs. However, both VEGF and bFGF transcripts increased after administration of PDGF-BB (10 or 20 ng/mL) or TGF-beta 1 (0.1 to 10 ng/mL). Hypoxia was a potent stimulus for VEGF gene expression but had no apparent effect on bFGF steady-state mRNA levels.

CONCLUSIONS

These results indicate that certain indirect angiogenic cytokines, such as PDGF-BB or TGF-beta 1, may act via induction of bFGF and VEGF gene expression in cells resident near endothelial cells in vivo. Hypoxia constitutes a potent stimulus for VEGF gene expresion but does not regulate bFGF under the same experimental conditions.

Through DNA microarray analysis and quantitative PCR verification, we have identified additional IL-17A-inducible genes-IL-19, CXCL-1, -2, -3, -5, and -6-in well-differentiated normal human bronchial epithelial cells. These genes, similar to previously described human beta-defensin-2 (HBD-2) and CCL-<em>20</em>, were induced by a basolateral treatment of IL-17A, and regulated by PI3K signaling and NF-kappaB activation. For PI3K signaling, increases of cellular PIP(3) and phosphorylation of downstream molecules, such as Akt and glycogen synthase kinase-3beta (GSK3beta) (S9), were detected. Induced gene expression and HBD-2 promoter activity were attenuated by LY294002, p110alpha small-interfering RNA (siRNA), as well as by an overexpression of constitutively active GSK3beta(S9A) or wild-type phosphatase and tensin homolog. Increased phosphorylation of JAK1/2 after IL-17A treatment was detected in primary normal human bronchial epithelium cells. Transfected siRNAs of JAK molecules and JAK inhibitor I decreased IL-17A-induced gene expression and GSK3beta(S9) phosphorylation. However, both JAK inhibitor I and PI3K inhibitor had no effect on the DNA-binding activities of p65 and p50 to NF-kappaB consensus sequences. This result suggested a JAK-associated PI3K signaling axis is independent from NF-kappaB activation. With siRNA to knockdown STIR (similar expression to <em>fibroblast</em> <em>growth</em> <em>factor</em> and IL-17R; Toll-IL-1R)-related signaling molecules, such as Act1, TNFR-associated <em>factor</em> 6 (TRAF6), and TGF-beta-activated kinase 1 (TAK1), and transfection of A52R, an inhibitor of the MyD88/TRAF6 complex, or dominant-negative TAK1, IL-17A-inducible gene expression and HBD-2 promoter activity were reduced. Additionally, IL-17A-induced p65 and p50 NF-kappaB activations were confirmed and their nuclear translocations were down-regulated by siRNAs of TRAF6 and TAK1. These results suggest that two independent and indispensable signaling pathways-1) JAK1-associated PI3K signaling and 2) Act1/TRAF6/TAK1-mediated NF-kappaB activation-are stimulated by IL-17A to regulate gene induction in human airway epithelial cells.

The underlying basis of many forms of syndromic craniosynostosis has been defined on a molecular level. However, many patients with familial or sporadic craniosynostosis do not have the classical findings of those craniosynostosis syndromes. Here we present 61 individuals from <em>20</em> unrelated families where coronal synostosis is due to an amino acid substitution (Pro250Arg) that results from a single point mutation in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 gene on chromosome 4p. In this instance, a new clinical syndrome is being defined on the basis of the molecular finding. In addition to the skull findings, some patients had abnormalities on radiographs of hands and feet, including thimble-like middle phalanges, coned epiphyses, and carpal and tarsal fusions. Brachydactyly was seen in some cases; none had clinically significant syndactyly or deviation of the great toe. Sensorineural hearing loss was present in some, and developmental delay was seen in a minority. While the radiological findings of hands and feet can be very helpful in diagnosing this syndrome, it is not in all cases clearly distinguishable on a clinical basis from other craniosynostosis syndromes. Therefore, this mutation should be tested for in patients with coronal synostosis.

Although epithelial-mesenchymal (stromal) interactions are thought to play an important role in embryonic and postnatal development of the mammary gland, the underlying mechanisms are still poorly understood. To address this issue, we assessed the effect of <em>fibroblast</em>-derived diffusible <em>factors</em> on the <em>growth</em> and morphogenetic properties of a clonally derived subpopulation (clone TAC-2) of normal murine mammary gland (NMuMG) epithelial cells embedded in collagen gels. Under control conditions, TAC-2 mammary gland epithelial cells suspended within collagen gels formed either irregularly shaped cell aggregates or short branching cord-like structures. Addition of conditioned medium from Swiss 3T3 or MRC-5 <em>fibroblasts</em> dramatically stimulated cord formation by TAC-2 cells, resulting in the development of an extensive, highly arborized system of duct-like structures, which in appropriate sections were seen to contain a central lumen. The effect of <em>fibroblast</em> conditioned medium was completely abrogated by antibodies against hepatocyte <em>growth</em> <em>factor</em> (also known as scatter <em>factor</em>), a <em>fibroblast</em>-derived polypeptide that we have previously shown induces tubulogenesis by Madin-Darby canine kidney epithelial cells. Addition of exogenous recombinant human hepatocyte <em>growth</em> <em>factor</em> to collagen gel cultures of TAC-2 cells mimicked the tubulogenic activity of <em>fibroblast</em> conditioned medium by stimulating formation of branching duct-like structures in a dose-dependent manner, with a maximal 77-fold increase in cord length at <em>20</em> ng/ml. The effect of either <em>fibroblast</em> conditioned medium or hepatocyte <em>growth</em> <em>factor</em> was markedly potentiated by the simultaneous addition of hydrocortisone (1 microgram/ml), which also enhanced lumen formation. These results demonstrate that hepatocyte <em>growth</em> <em>factor</em> promotes the formation of branching duct-like structures by mammary gland epithelial cells in vitro, and suggest that it may act as a mediator of the inducing effect of mesenchyme (or stroma) on mammary gland development.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF <em>fibroblasts</em> produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03-2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin(-/-)) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-<em>20</em> Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin(-/-) mice were protected from bleomycin-induced fibrosis. Instillation of OC-<em>20</em> during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming <em>growth</em> <em>factor</em>-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention.

Exuberant tumor-like synovial cell proliferation with invasion of periarticular bone is a feature of rheumatoid arthritis in humans and of streptococcal cell wall (SCW)-induced arthritis in rats. These histologic observations prompted us to examine synoviocytes from arthritic joints for phenotypic characteristics of transformed cells. The capacity to grow in vitro under anchorage-independent conditions is a characteristic that correlates closely with potential in vivo tumorigenicity. In medium supplemented with <em>20</em>% serum or in basal media supplemented with platelet-derived <em>growth</em> <em>factor</em> (PDGF), early passage synoviocytes from both SCW-induced and rheumatoid arthritic joints formed colonies in soft agarose. Epidermal <em>growth</em> <em>factor</em> (EGF), interleukin 1 (IL-1), tumor necrosis <em>factor</em>-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) did not support <em>growth</em>, although EGF enhanced PDGF-dependent <em>growth</em>. On the other hand, TGF-beta, as well as all-trans-retinoic acid, inhibited colony <em>growth</em>. Early passage normal rat and human synoviocytes also grew under the same conditions, but lung, skin, and late-gestation embryonic <em>fibroblast</em>-like cells did not. Considered in the context of other published data our findings provide cogent evidence that synoviocytes, but not other types of <em>fibroblast</em>-like cells, readily acquire phenotypic characteristics commonly associated with transformed cells. Expression of the transformed phenotype in the inflammatory site is likely regulated by paracrine <em>growth</em> <em>factors</em>, such as PDGF and TGF-beta.