BACKGROUND

The highly vascular nature of renal cell carcinoma (RCC) suggests that angiogenesis inhibition may be therapeutic for patients with this disease. Thalidomide inhibits basic fibroblast growth factor and vascular endothelial growth factor (VEGF)-induced angiogenesis.

METHODS

In a pilot study, we evaluated the safety and efficacy of escalating doses of thalidomide in patients with progressive metastatic RCC (mRCC), measurable disease, and good organ function. Patients received oral thalidomide starting at 200 mg per day and increasing by 100-200 mg per day weekly until a target dose of 1200 mg per day was reached. Study endpoints were objective tumor response and toxicity.

RESULTS

Of the 20 patients enrolled, 19 were evaluable for response. Eighteen achieved the target dose. The most common, but reversible, toxicities were constipation, somnolence, and fatigue. Peripheral neuropathy was seen after prolonged therapy, necessitating dose reduction. Two patients achieved a partial response and nine had stable disease for a median of 14 months (range, 3-17 months). Median time to progression was 4.7 months (range, 0.7-31.3 months). Fifteen patients died (median survival, 18.1 months; 95% lower confidence bound 10.7). Survival was significantly longer in patients with higher hemoglobin level and longer time from first metastasis to start of thalidomide, but significantly shorter in patients with multiple organ involvement and previous treatments.

CONCLUSIONS

Thalidomide at this dose is associated with manageable acute toxicities but long-term dose-limiting neuropathy. Objective responses are rare in patients with mRCC and are characterized by delay in achieving maximum tumor reduction. Prolonged stable disease is seen in some patients, but the benefit of thalidomide, as well as other angiogenesis inhibitors, in that setting needs to be studied in controlled, randomized trials.

A series of peptides, termed dekafins, were derived from the beta10-beta11 loop regions of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) 1, 2, 3, 5, 6, 8, 9, 10, and <em>17</em>. The dekafins share a homologous amino acid sequence similar to a sequence in the first fibronectin type III module of the neural cell adhesion molecule. All dekafins were shown by surface plasmon resonance analysis to bind <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR)1-IIIc-Ig2-3 and FGFR2-IIIb-Ig2-3, respectively, with K(d) values of approximately 10(-7) to 10(-8) mol/L. Binding of dekafin1 to FGFR1-IIIc-Ig2-3 was inhibited by a heparin analog, sucrose octasulfate, indicating that heparin sulfate moiety can modulate dekafin binding to FGFRs. Treatment of transcription and mRNA export (TREX) cells permanently expressing Strep-tag-labeled FGFR1-IIIc with dekafins resulted in receptor phosphorylation. FGF1-induced FGFR1-IIIc phosphorylation was inhibited by dekafin1 and 10 in high concentrations, indicating that dekafins are FGFR partial agonists. The dekafins induced neuronal differentiation as reflected by neurite out<em>growth</em> from cerebellar granule neurons, an effect that was abolished by SU5402, a specific inhibitor of the FGFR tyrosine kinase, and by inositolhexaphosphate, an extracellularly acting FGFR antagonist. Some, but not all, dekafins were capable of promoting survival of cerebellar granule neurons induced to undergo apoptosis. Thus, the dekafins are functional FGFR agonists with apparent therapeutic potential.

<AbstractText>The objective of this study was to measure changes in glucose, lipid, and inflammation parameters after transitioning from a baseline diet (BD) to an isocaloric ketogenic diet (KD).</AbstractText><p><div><b>METHODS</b></div>Glucose homeostasis, lipid homeostasis, and inflammation were studied in <em>17</em> men (BMI: 25-35 kg/m² ) during 4 weeks of a BD (15% protein, 50% carbohydrate, 35% fat) followed by 4 weeks of an isocaloric KD (15% protein, 5% carbohydrate, 80% fat). Postprandial responses were assessed following mixed-meal tests matched to compositions of the BD (control meal [CM]) and KD (ketogenic meal).</p><AbstractText>Fasting ketones, glycerol, free fatty acids, glucagon, adiponectin, gastric inhibitory peptide, total and low-density lipoprotein cholesterol, and C-reactive protein were significantly increased on the KD. Fasting insulin, C-peptides, triglycerides, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 were significantly decreased. During the KD, the glucose area under the curve was significantly higher with both test meals, and the insulin area under the curve was significantly higher only for the CM. Analyses of glucose homeostasis suggested that the KD insulin sensitivity decreased during the CM but increased during the ketogenic meal. Insulin-mediated antilipolysis was decreased on the KD regardless of meal type.</AbstractText><AbstractText>Switching to the KD was associated with increased cholesterol and inflammatory markers, decreased triglycerides, and decreased insulin-mediated antilipolysis. Glucose homeostasis parameters were diet dependent and test meal dependent.</AbstractText>

Phosphaturic mesenchymal tumor, mixed connective tissue type (PMTMCT) is a rare neoplasm that can cause tumor-induced osteomalacia due to overproduction of a phosphaturic hormone, <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23). We report here a case of subcutaneous PMTMCT, non-phosphaturic variant, in the sole. We also review 32 Japanese cases of PMTMCT reported in detail. They occurred in 16 men and 15 women (one was unknown), with ages ranging 20-73 years (median, 48). Tumors were found in soft tissue, bone and sinuses in <em>17</em>, 11 and four, respectively. A history of long-standing osteomalacia was noted in all cases except two non-phosphaturic variant cases. Serum FGF23 level was elevated in 11 of 12 cases examined. In terms of follow-up information, metastases were found in four patients, and two patients died of disease. In conclusion, PMTMCT is histologically a benign lesion; however, there may be rare metastatic and malignant cases. Wider recognition of the histological features of this unique neoplasm would aid its distinction from the large number of mesenchymal tumors for which it may be mistaken and should enable correct diagnosis of tumors with osteomalacia.

Mortality associated with breast cancer is attributable to aggressive metastasis, to which TNFα plays a central orchestrating role. TNFα acts on breast tumor TNF receptors evoking the release of chemotactic proteins (e.g. MCP-1/CCL2). These proteins direct inward infiltration/migration of tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), T-regulatory cells (Tregs), T helper IL-<em>17</em>-producing cells (Th<em>17</em>s), metastasis-associated macrophages (MAMs) and cancer-associated <em>fibroblasts</em> (CAFs). Tumor embedded infiltrates collectively enable immune evasion, tumor <em>growth</em>, angiogenesis, and metastasis. In the current study, we investigate the potential of apigenin, a known anti-inflammatory constituent of parsley, to downregulate TNFα mediated release of chemokines from human triple-negative cells (MDA-MB-231 cells). The results show that TNFα stimulation leads to large rise of CCL2, granulocyte macrophage colony-stimulating <em>factor</em> (GMCSF), IL-1α and IL-6, all suppressed by apigenin. While many aspects of the transcriptome for NFkB signaling were evaluated, the data show signaling patterns associated with CCL2 were blocked by apigenin and mediated through suppressed mRNA and protein synthesis of IKBKe. Moreover, the data show that the attenuation of CCL2 by apigenin in the presence TNFα paralleled the suppression of phosphorylated extracellular signal-regulated kinase 1 (ERK 1/ 2). In summary, the obtained findings suggest that there exists a TNFα evoked release of CCL2 and other LSP recruiting cytokines from human breast cancer cells, which can be attenuated by apigenin.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte <em>growth</em> <em>factor</em> (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin <em>17</em> compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a <em>fibroblast</em> feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

MicroRNAs (miRNAs) play important roles in the fibrosis of systemic sclerosis (SSc). However, the underlying miRNA-mRNA regulatory network is not fully understood. A systemic investigation of the role of miRNAs would be very valuable for increasing our knowledge of the pathogenesis of SSc. Here, we combined miRNA and mRNA expression profiles and bioinformatics analyses and then performed validation experiments. we identified 21 miRNAs and 2698 mRNAs that were differentially expressed in SSc. Among these, <em>17</em> miRNAs and their 33 target mRNAs (55 miRNA-mRNA pairs) were involved in Toll-like receptor, transforming <em>growth</em> <em>factor</em> β and Wnt signalling pathways. Validation experiments revealed that miR-146b, miR-130b, miR-21, miR-31 and miR-34a levels were higher whereas miR-145 levels were lower in SSc skin tissues and <em>fibroblasts</em>, normal <em>fibroblasts</em> and endothelial cells that were stimulated with SSc serum. ACVR2B, FZD2, FZD5 and SOX2 levels were increased in SSc skin <em>fibroblasts</em>, normal <em>fibroblasts</em> and endothelial cells that were stimulated with SSc serum. We did not identify any negative correlations among these miRNA-mRNA pairs. miR-21 was specifically expressed at higher levels in SSc serum. Six miRNAs and 4 mRNAs appear to play important roles in the pathogenesis of SSc are worth investigating in future functional studies.

The Na-K-2Cl cotransporter NKCC1 is an important volume-regulatory transporter that is regulated by cell volume and intracellular Cl(-). This regulation appears to be mediated by phosphorylation of NKCC1, although there is evidence for additional, cytoskeletal regulation via myosin light chain (MLC) kinase. NKCC1 is also activated by <em>growth</em> <em>factors</em> and may contribute to cell hypertrophy, but the mechanism is unknown. In aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive (86)Rb(+) influx) was rapidly stimulated by serum, lysophosphatidic acid, and <em>fibroblast</em> <em>growth</em> <em>factor</em>, with the greatest stimulation by serum. Serum increased bumetanide-sensitive influx significantly more than bumetanide-sensitive efflux (131% vs. 44%), indicating asymmetric stimulation of NKCC1, and produced a <em>17</em>% increase in cell volume and a 25% increase in Cl(-) content over 15 min. Stimulation by serum and hypertonic shrinkage were additive, and serum did not increase phosphorylation of NKCC1 or MLC, and did not decrease cellular Cl(-) content. When cellular Cl(-) was replaced with methanesulfonate, influx via NKCC1 increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose a novel mechanism whereby serum activates NKCC1 by reducing its sensitivity to inhibition by intracellular Cl(-). This resetting of the Cl(-) set point of the transporter enables the cotransporter to produce a hypertrophic volume increase.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are cell mitogens and differentiating <em>factors</em> with neuroprotective properties in the CNS. We have already shown that endothelial cells genetically engineered to secrete human FGF-1 (RBEZ-FGF) survive implantation to neonatal rat brain (Johnston et al. (1996) J. Neurochem. 67, 1643-1652]. In this study, the effects of cell-based FGF-1 gene delivery on quinolinate-induced neurotoxicity in the developing rat brain were examined. Control endothelial cells (RBE4), and RBEZ-FGF cells were implanted into right striatum at post-natal day (PND) 7. On PND 10, quinolinate (150 nmol), an endogenous N-methyl-d-aspartate (NMDA) receptor agonist, or vehicle alone was injected into striatum ipsilateral to cell implantation. Injury was quantified in coronal sections obtained from PND <em>17</em> animals by comparing striatal and hippocampal volumes ipsilateral and contralateral to the site of quinolinate injection. Human FGF-1 specific transgene expression in vivo was shown by Northern blot and RT-PCR up to 14 days after cell implantation in control animals, and up to 4 days after quinolinate exposure. Quinolinate reduced the size of ipsilateral striatum by 37% and hippocampus by 38% in animals preimplanted with control endothelial cells. In contrast, quinolinate reduced the size of striatum by only 14% and had no effect on hippocampal size in animals preimplanted with RBEZ-FGF cells. Thus, FGF-1 gene delivery protected the developing striatum and hippocampus from quinolinate-induced volume loss by 62% and 100%, respectively. Intrastriatal quinolinate resulted in a significant decrease in density of NOS+ CA3 hippocampal neurons (-38%) without affecting the density of NOS+ neurons in hippocampal regions CA1, dentate gyrus or striatum. This response of CA3 NOS+ neurons appeared to be only partially reversed by FGF-1 gene delivery. Our results show that intracerebral FGF-1 gene expression within the developing brain can protect striatum and hippocampus from quinolinate-mediated injury.

The components of the extracellular signal-regulated kinase (ERK) pathway are involved in the regulation of epididymal cellular processes. Interestingly, our previous studies showed that there are two different activity levels of the ERK pathway components in the epididymal epithelium: a basal level in most regions and a higher level in the differentiated initial segment (IS). In this study we analyzed the role of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor substrate 2 (FRS2) in the regulation of these two levels. Two mouse models were generated. In the first model, Frs2 was deleted from epithelial cells of most epididymal regions except for the IS from the embryonic period onward. Loss of Frs2 dampened the basal activity level of the ERK pathway components, which resulted in an increase in apoptosis along the epididymal duct. This was observed during the period when FRS2 expression level was highest in wild-type epididymides. In the second model, Frs2 was deleted from the proximal epididymal epithelium from Postnatal Day <em>17</em> onward. Most of the epididymides in this model exhibited normal morphology. Loss of Frs2 in these epididymides did not affect the high activity level of the ERK pathway components in the IS. However, a subgroup of epididymides in the second model showed increased apoptosis which resulted in an abnormally shaped proximal region or development of granulomas. Therefore, data from these two models showed that FRS2 played different roles in the regulation of two activity levels of the ERK pathway components in the epididymis.

IL-<em>17</em> cytokines play a crucial role in a variety of inflammatory and autoimmune diseases. They signal through heterodimeric receptor complexes consisting of members of IL-<em>17</em>R family. A unique intracellular signaling domain was identified within all IL-<em>17</em>Rs, termed similar expression to <em>fibroblast</em> <em>growth</em> <em>factor</em> genes and IL-<em>17</em>R (SEFIR). SEFIR is also found in NF-κB activator 1 (Act1), an E3 ubiquitin ligase, and mediates its recruitment to IL-<em>17</em>Rs. In this study, to our knowledge, we report the structure of the first SEFIR domain from IL-<em>17</em>RB at 1.8Å resolution. SEFIR displays a five-stranded parallel β-sheet that is wrapped by six helices. Site-directed mutagenesis on IL-<em>17</em>RB identified helix αC as being critical for its interaction with Act1 and IL-25 (IL-<em>17</em>E) signaling. Using the current SEFIR structure as a template, the key functional residues in Act1 are also mapped as part of helix αC, which is conserved in IL-<em>17</em>RA and RC, suggesting this helix as a common structural signature for heterotypic SEFIR-SEFIR association. In contrast, helix αB' is important for homodimerization of Act1, implicating a dual ligand-binding model for SEFIR domain, with distinct structural motifs participating in either homotypic or heterotypic interactions. Furthermore, although the IL-<em>17</em>RB-SEFIR structure resembles closest to the Toll/IL-1R domain of TLR10 with low sequence homology, substantial differences were observed at helices αC, αD, and DD' loop. To our knowledge, this study provides the first structural view of the IL-<em>17</em>R intracellular signaling, unraveling the mechanism for the specificity of SEFIR versus Toll/IL-1R domain in their respective signaling pathways.

Cellular interactions between stroma and epithelium are important in the <em>growth</em> and proliferation of prostate cancer. Peptide <em>growth</em> <em>factors</em> may facilitate the progression of prostate cancer as autocrine and/or paracrine <em>factors</em>. Keratinocyte <em>Growth</em> <em>Factor</em> (KGF or FGF7) has a differentiative and proliferative effect on the epithelium of the developing rat prostate. We investigated if KGF may act as a paracrine agent in human prostate cancer and examined the expression of KGF and <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> Receptors (FGFRs) (IIIb and IIIc isoforms of the FGFR1 and FGFR2 genes). Sixty-five percent (11 out of <em>17</em> informative cases) of prostate cancers (CaP) expressed KGF mRNA by RT-PCR, while KGF expression was not detected in benign prostatic hyperplasia (BPH) (n = 6). Upregulation of KGF expression was related to hormone insensitive tumours (P<0.05). Tumour grade and stage were not associated with KGF expression. The source of KGF expression was further characterised using an in vitro primary culture model, showing its restriction to the prostatic stroma. The FGFR1IIIb isoform was expressed in all cases of prostate cancer (n = <em>17</em>), and FGFR1IIIc mRNA was not detected. In the BPH group, FGFR1IIIb transcripts were detected in four out of six cases. FGFR2IIIb expression was detected in five of six cases of BPH and twelve out of seventeen (71%) cases of prostate cancer. In CaP, though not reaching statistical significance, the persistence of FGFR2IIIb expression appeared to be associated with hormone insensitive tumours (P=0.052). FGFR2IIIc expression was present in eleven of seventeen tumours but was absent in all six cases of BPH. Functional assessment of recombinant KGF in a proliferation assay demonstrated a mitogenic effect of up to 100% on cultured prostatic epithelial cells.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming <em>growth</em> <em>factor</em>-β1 (TGFβ1) to induce <em>fibroblast</em>ic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into <em>fibroblast</em>ic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-<em>17</em>. Exposure to bFGF for 2 days promoted vascular endothelial <em>growth</em> <em>factor</em> gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

OBJECTIVE

The selection of a suitable cell type for scaffold seeding, its isolation and adequate expansion in vitro remains a major challenge in tissue valve engineering. The study aim was to establish a model for efficient procurement of myofibroblasts for in-vitro seeding using fibroblasts as progenitor cells.

METHODS

Dermal and arterial mesenchymal cells from human (hDMC1.1 and hAMC1.1) and sheep (sDMC1.1 and sAMC1.1) were isolated by sequential culture. Cell isolates were characterized by stringent criteria based on morphology, immunocytochemistry using antibodies to vimentin, cytokeratin, prolyl 4-hydroxylase, smooth muscle alpha-actin (alpha-SMA) and smooth muscle myosin, and by Western blotting for alpha-SMA and N-cadherin. Cultures with less than 10-20% alpha-SMA-positive cells were considered to be fibroblastic. Cells were subsequently transdifferentiated with the cytokine transforming growth factor-beta1 (TGF-beta1) during five days, and then evaluated morphotypically, by immunocytochemistry, and by Western blotting. The metabolic and functional properties of TGF-beta1-treated and untreated colonies were compared by measuring the expression of extracellular proteins (collagen type 1 and tenascin-C) and by a collagen matrix contraction assay.

RESULTS

TGF-beta1 successfully transformed both human and sheep fibroblasts to metabolically active and functional myofibroblasts based on stringent criteria for myofibroblast characterization. Alpha-SMA positivity of 100% was obtained in all cases (hDMC1.1, hAMC1.1, sDMC1.1, and sAMC1.1) after transformation compared to less than 50% in the non-transformed state (hAMC1.1, 17%; hDMC1.1, 10%; sAMC1.1, 43%; and sDMC1.1, 30%). This observation was further supported by increased contractility and an up-regulation of extracellular protein production in transdifferentiated cells.

CONCLUSIONS

Untreated arterial cell isolates were, at best, less than 50% alpha-SMA-positive. By allowing procurement of high densities of myofibroblasts in a relatively short time, the model was seen to be a potentially useful tool in tissue valve engineering, at least in investigations using autologous cells in the sheep model.

Loss of intestinal CD4(+) T cells was associated with decreased production of several T-helper 1 (TH1) and TH2 cytokines and increased production of interleukin <em>17</em> (IL-<em>17</em>), gamma interferon (IFN-γ), CCL4, and granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) by CD8(+) T cells 21 days after simian immunodeficiency virus (SIV) infection in rhesus macaques. Shifting of mucosal TH1 to TH2 or T-cytotoxic 1 (TC1) to TC2 cytokine profiles was not evident. Additionally, both CD4(+) and CD8(+) T cells showed upregulation of macrophage migration inhibition <em>factor</em> (MIF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-basic) cytokines that have been linked to HIV disease progression.

Achondroplasia (ACH), the most common cause of chondrodysplasia in man (1 in 15,000 live births), is an autosomal dominant condition of unknown origin characterized by short-limbed dwarfism and macrocephaly. Recently, a gene for ACH has been mapped to chromosome 4p16.3. The genetic interval encompassing the disease gene contains a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) family which is expressed in articular chondrocytes (FGFR3). We report here recurrent missense mutations, in a CpG doublet of the transmembrane domain of the FGFR3 protein (G380R) in <em>17</em> sporadic cases and 6 unrelated familial forms of ACH and show that the mutant genotype segregates with the disease in these families. Thus, it appears that recurrent mutations of a single amino acid in the transmembrane domain of the FGFR3 protein account for all cases (23/23) of achondroplasia in our series.

The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin, DMRIE-C, DAC-30, Effectene, FuGene6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity (n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (<em>17</em>+/-2.02%) transfected HCEC more successfully than DMRIE-C (11+/-1.46%), Effectene (9+/-0.62%), FuGene (9+/-0.93%) and DAC-30 (7+/-0.59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 (P<0.05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.

Interaction of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR) sufficient for a trans-phosphorylation event in which one intracellular domain is substrate for the other is essential for signal transduction. By analysis of the direct interaction of recombinant constructions co-expressed in baculoviral-infected insect cells, we identified a <em>17</em>-amino acid sequence that is required for the stable interaction between ectodomains of FGFR. The sequence 160ERSPHRPILQAGLPANK<em>17</em>6 (Glu160-Lys<em>17</em>6) connects immunoglobulin modules II and III. In insect cells, the interaction between Glu160-Lys<em>17</em>6 domains occurs independently of intact heparin or FGF binding domains. The sequence is not required for the binding of heparin or FGF-1, but is essential for mitogenic activity of the FGFR kinase in mammalian cells. The results support a model in which the homeo-interaction between Glu160-Lys<em>17</em>6 in the ectodomain contributes to the interaction between intracellular domains in mammalian cell membranes (Kan, M., Wang, F., Kan, M., To, B., Gabriel, J. L., and McKeehan, W. L. (1996) J. Biol. Chem. 271, 26143-26148). We propose that the Glu160-Lys<em>17</em>6 domain plays a pivotal role in restriction of the interaction between kinases by pericellular matrix heparan sulfate proteoglycan and divalent cations. Restrictions are overcome by FGF or constitutively by diverse gain of function mutations which cause skeletal and craniofacial abnormalities.

OBJECTIVE

Type 2 diabetes is often complicated by diabetic foot syndrome (DFS). We analyzed the circulating stem cells, growth factor and anti-oxidant gene expression profiles in type 2 diabetes patients without or with different forms of DFS.

METHODS

Healthy volunteers (n = 13) and type 2 diabetes patients: (i) without DFS (n = 10); or with (ii) Charcot osteoneuropathy (n = 10); (iii) non-infected (n = 17); (iv) infected (n = 11); and (v) healed ulceration were examined (n = 12). Peripheral blood endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and very small embryonic-like (VSEL) cells were phenotyped using flow cytometry. Plasma cytokine concentrations and gene expressions in blood cells were measured by Luminex and quantitative real-time polymerase chain reaction assays, respectively.

RESULTS

Patients with non-complicated type 2 diabetes showed reduced HMOX1 expression, accompanied by HMOX2 upregulation, and had less circulating EPC, MSC or HSC than healthy subjects. In contrast, VSEL cells were elevated in the type 2 diabetes group. However, subjects with DFS, even with healed ulceration, had fewer VSEL cells, more CD45-CD29(+)CD90(+)MSC, and upregulated HMOX1 when compared with the type 2 diabetes group. Patients with Charcot osteopathy had lowered plasma fibroblast growth factor-2. Elevated plasma tumor necrosis factor-α and decreased catalase expression was found in all diabetic patients.

CONCLUSIONS

Patients with type 2 diabetes and different forms of DFS have an altered number of circulating stem cells. Type 2 diabetes might also be associated with a changed plasma growth factor and anti-oxidant gene expression profile. Altogether, these factors could contribute to the pathogenesis of different forms of DFS.

Most patients with severe coronary artery disease have normal baseline myocardial blood flow. Therefore, interventions aimed at inducing therapeutic angiogenesis in these patients should cause new blood vessel <em>growth</em> in the heart in the absence of chronic ischemia. It was examined whether adenovirus-mediated gene transfer of recombinant, secreted acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (sp+aFGF(1-154)), next to a major epicardial artery, may induce neovascularization and reduce the risk region for myocardial infarction upon coronary ligation near the injection site. Fifteen days prior to coronary artery occlusion, rabbits were treated with intramyocardial injections of AdCMV.sp+aFGF(1-154), the control vector AdCMV.NLSbetagal (1 x 10(9) plaque-forming units), or saline. Messenger RNA transcripts for aFGF(1-154) were present up to 12 days after injection in the tissues exposed to AdCMV.aFGF(1-154). Following coronary artery occlusion rabbits treated with AdCMV. sp+aFGF(1-154) showed a 50% reduction of the risk region for myocardial infarction (P < 0.01 vs control). Histologic analysis showed a twofold increase in length density of intramural coronary arterioles (P < 0.01 vs control) and a <em>17</em>% increase in length density of the capillary network (P < 0.001) in the myocardium exposed to AdCMV.sp+aFGF(1-154). Thus, gene therapy with AdCMV. sp+aFGF(1-154) can induce angiogenesis in the absence of chronic ischemia. The newly formed collateral blood vessels provide an anatomical basis for the reduction in the risk region for myocardial infarction upon subsequent occlusion of the coronary artery in proximity of the site where angiogenesis was induced.

Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein, found almost exclusively in mineralized connective tissues, that may function in the nucleation of hydroxyapatite crystals. We have found that expression of BSP in osteoblastic ROS <em>17</em>/2.8 cells is stimulated by <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), a potent mitogen for mesenchymal cells. Stimulation of BSP mRNA with 10 ng/ml FGF2 was first evident at 3 h ( approximately 2.6-fold) and reached maximal levels at 6 h ( approximately 4-fold). From transient transfection assays, a FGF response element (FRE) was identified (nucleotides -92 to -85, "GGTGAGAA") as a target of transcriptional activation by FGF2. Ligation of two copies of the FRE 5' to an SV40 promoter was sufficient to confer FGF-responsive transcription. A sequence-specific protein-DNA complex, formed with a double-stranded oligonucleotide encompassing the FRE and nuclear extracts from ROS <em>17</em>/2.8 cells, but not from <em>fibroblasts</em>, was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed both basal and FGF2-mediated promoter activity. These studies, therefore, have identified a novel FRE in the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription.

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin <em>17</em> (IL-<em>17</em>) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), on BM-MSCs <em>growth</em> and elucidated the signaling pathways involved. The results revealed that IL-<em>17</em> increased the frequency of colony-forming units <em>fibroblast</em> (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-<em>17</em> and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-<em>17</em> and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.

Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation <em>factor</em> promoter. The pEFNeo expression vector also contains the human <em>growth</em> hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.<em>17</em>, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell <em>factor</em>ies. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcFlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by <em>fibroblasts</em> from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.

Human osteoclasts are well characterized multinucleated cells whose function is the directed resorption of normal bone (NB). Osteoclastic bone destruction accompanies lytic solid tumors and myeloma as well as Paget's disease (PD) of bone and giant cell tumors of bone (GCTB). The mechanism of this stimulation of osteoclastic bone resorption is unknown. This study was designed to detect cytokines present in the multinucleated cells of PD and GCTB in order to determine whether cytokine abnormalities exist to account for bone lysis. Nine cytokines, representing the functions of bone resorption, angiogenesis, tumor necrosis, bone cell proliferation, and osteoblast-osteoclast coupling, were examined by immunohistochemistry using tissue samples from 15 NB, <em>17</em> PD, and 19 GCTB patients. Standard nonparametric statistical analysis showed a significant increase (P < 0.01 to 0.05) in immunostaining between osteoclasts of PD and NB for interleukin-6 (Il-6), tumor necrosis <em>factor</em> beta (TNFbeta), epidermal <em>growth</em> <em>factor</em> (EGF), platelet derived <em>growth</em> <em>factor</em> (PDGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). There was a statistically significant decrease in immunostaining of giant cells of GCTB as compared with NB for transforming <em>growth</em> <em>factor</em> beta (TGFbeta), but no other differences from normal osteoclasts. The increase in staining of PD osteoclasts over the giant cells of GCTB was significant (P < 0.01) for Il-6, TNFbeta, PDGF, bFGF and insulin <em>growth</em> <em>factor</em>-1 (IGF-1), and (P < 0. 05) for Il-1 and EGF. It was concluded that marked cytokine differences exist in vivo between osteoclasts of NB and PD lesions consistent with stimulated resorption. Alternatively, "osteoclastoma" cells in the center of the tumor did not overexpress the cytokines associated with bone lysis, suggesting some other mechanism for stimulated resorption.