OBJECTIVE

The effects of cell transplantation on the ischemic failing heart have already been documented. However, the area in and around infarct regions is not a good environment for cells to survive in because they are exposed to poor conditions in which certain requirements cannot be adequately supplied. We therefore designed a study to investigate the efficacy of prevascularization in ischemic regions before cell transplantation.

METHODS

Rats with myocardial infarction were randomized into 4 groups: 11 rats received a culture medium injection to the left ventricular wall (control group), 11 received fetal cardiomyocyte transplantation (TX group), 11 received gelatin hydrogel microspheres incorporating basic fibroblast growth factor (FGF group), and 11 received basic fibroblast growth factor pretreatment sequentially, followed by cardiomyocyte transplantation (FGF-TX group). Four weeks later, left ventricular function was assessed by means of echocardiography and cardiac catheterization.

RESULTS

In the FGF and FGF-TX groups neovascularization was found in the scar tissue 1 week later. The TX, FGF, and FGF-TX groups showed better fractional shortening than the control group (TX, FGF, FGF-TX, and control: 28% +/- 4.4%, 24% +/- 8.6%, 27% +/- 7.3%, and 17% +/- 4.6%, respectively; P <.01). Left ventricular maximum time-varying elastance was higher in the FGF-TX group than in the TX and FGF groups (FGF-TX, TX, and FGF: 0.52 +/- 0.23, 0.30 +/- 0.08, and 0.27 +/- 0.20 mm Hg/microL, respectively; P <.01). Histologically, more transplanted cells survived in the FGF-TX group than in the TX group.

CONCLUSIONS

Prevascularization with basic fibroblast growth factor-incorporated microspheres enhances the benefits of cardiomyocyte transplantation. We expect that this system will contribute to regeneration medicine through its extensive application to other growth factors.

Nucleotide sequencing of the two known cDNAs encoding human insulin-like <em>growth</em> <em>factor</em> I (IGF-I) predicts the existence of two different prohormone forms of IGF-I. The E peptide regions extend the carboxy-terminus of IGF-I by either an additional 35 (IGF-IA) or 77 (IGF-IB) amino acids. With a specific and sensitive RIA employing an antiserum directed against a synthetic peptide that is unique to the E peptide region of IGF-IA prohormone (EIA), we have identified EIA-immunoreactive material in the conditioned medium of fetal and postnatal human <em>fibroblasts</em> in culture. Incubation of postnatal human <em>fibroblasts</em> with GH increased specific immunoreactive EIA secretion 2- to 3-fold. There was no immunologically detectable 7.5K IGF-I or IGF-II peptide in acid-chromatographed human <em>fibroblast</em>-conditioned medium under either basal or GH-stimulated conditions. Acid chromatography of human <em>fibroblast</em>-conditioned medium on Sephadex G-75 revealed a single elution peak of EIA immunoreactivity corresponding to a mol wt of 9-<em>17</em> K. With neutral chromatography, EIA immunoreactivity eluted at 25-38K mol wt. These data suggest that the E peptide region of IGF-IA is translated and released as part of the prohormone form in cultured human <em>fibroblasts</em>, and that the levels of this prohormone are regulated by GH.

OBJECTIVE

Matrilysin, matrix metalloproteinase (MMP)-7, is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. The purpose of this study was to determine the role of matrilysin in maintaining corneal avascularity during wound healing.

METHODS

Matrilysin-deficient mice (n = <em>17</em>) and their age-matched wild-type littermates (n = 18) were treated with 193 nm argon-fluoride excimer keratectomy (experiment I). The percentage of corneal surface occupied by neovascularization was measured with a computer image-analysis program adjusted for parallax. In another experiment (experiment II), epithelial closure was monitored with slit lamp biomicroscopy and fluorescein staining, and corneal neovascularization was confirmed by india ink perfusion, electron microscopy, and immunolocalization of CD31 and type IV collagen. Corneal micropocket assays were performed to compare the area of corneal neovascularization in matrilysin-deficient mice and wild-type littermates (experiment III). To determine whether the differences in corneal neovascularization were related to differences in angiogenic <em>factors</em>, the levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were compared with those of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in matrilysin-deficient and wild-type mouse corneas (experiment IV).

RESULTS

The percentages of the corneal surface occupied by neovascularization after excimer laser keratectomy in the matrilysin-deficient mice measured 21.3% +/- 5.2% and 18.7% +/- 5.8% at days 3 and 7, respectively, compared with 5.3% +/- 2.4% and 5.5% +/- 3.4% in the wild-type littermates at days 3 (P < 0.01) and 7, respectively (P < 0.05; experiment I). No significant differences in the rates of epithelial closure of corneal wounds were observed between matrilysin-deficient and wild-type mice after wounding. Corneal neovascularization in the matrilysin-deficient mice was confirmed by india ink present in the corneal stromal blood vessels (extending from the limbus to the wound), immunohistochemical staining, and electron microscopy. Gram, Giemsa, calcofluor white, and acridine orange stains and electron microscopy showed no evidence of corneal infection (experiment II). The area of corneal neovascularization in matrilysin-deficient mice was not significantly different from that of wild-type littermates after implantation of bFGF pellets (0.91 +/- 0.55 mm(2) and 0.77 +/- 0.34 mm(2), respectively; experiment III). The levels of bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) in corneal epithelial cells were not elevated in matrilysin-deficient mice compared with the wild-type mice (experiment IV).

CONCLUSIONS

Matrilysin may play an important role in maintaining corneal avascularity during wound healing. The differences in corneal neovascularization between matrilysin-deficient mice and wild-type littermates seem unrelated to the bFGF and VEGF levels in the corneal epithelium.

The role of female hormones in the prevalence of cardiac diseases are recognized but not fully explored. Proliferation of cardiac <em>fibroblasts</em>, the cellular origin of the extracellular matrix proteins, <em>growth</em> <em>factors</em> and cytokines in the heart, is an important underlying mechanism in the pathophysiological remodeling of the myocardium. In this study, we have investigated the effect of estrogen (<em>17</em> beta-estradiol) on proliferative capacity of cardiac <em>fibroblasts</em> obtained from adult female rat heart. DNA synthesis, as determined by incorporation of 3H-thymidine into DNA, increased in estrogen-treated cells. In the presence of tamoxifen, an anti-estrogen with high affinity for estrogen receptor. <em>17</em> beta-estradiol-induced stimulation of DNA synthesis was abolished. Alpha-estradiol, a stereo-isomer which does not bind the estrogen receptor, did not change DNA synthesis. In the presence of a synthetic inhibitor of MAP kinase pathway. PD98059, estrogen failed to stimulate DNA synthesis. In-gel kinase assays showed rapid and transient increased phosphorylation of MAP kinase substrate, myelin basic protein (MBP), at 42 and 44 kDa by <em>17</em> beta-estradiol, which was not observed in the presence of PD98059 and tamoxifen, not induced by alpha-estradiol and persisted in the absence of protein kinase C. In vitro kinase assay confirmed <em>17</em> beta-estradiol-induced activation of ERK1 and ERK2, with predominant effect on ERK2 in cardiac <em>fibroblasts</em>. The results of immunofluorescent light microscopy using anti-type alpha and beta estrogen receptor antibodies showed the expression of estrogen receptor types alpha and beta in control untreated cells, and indicated that type beta receptor is the predominant type with both cytoplasmic and nuclear localization. <em>17</em> beta-estradiol treatment of cardiac <em>fibroblasts</em> induced the translocation of receptor protein to the nuclei. Together, these data provide evidence that cardiac <em>fibroblasts</em> are cellular targets for direct effects of estrogen, and that this hormone enhances proliferative capacity of cardiac <em>fibroblasts</em> via estrogen receptor- and MAP kinase-dependent mechanisms. These data further suggest that estrogen, by its <em>growth</em>-enhancing effects in cardiac <em>fibroblasts</em>, can regulate the remodeling of the extracellular matrix and alter the microenvironment of cardiac cells, and hence exert an impact on the integrity of myocardial function.

Quantification of 27 cytokines following cerebral wounding was performed for wound age estimation. The cytokines evaluated included interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 p40, IL-12 p70, IL-15, IL-<em>17</em>, IL-18, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), granulocyte-colony stimulating <em>factor</em> (G-CSF), granulocyte macrophage-colony stimulating <em>factor</em> (GM-CSF), Interferon-gamma (IFN-gamma), keratinocyte derived cytokine (KC), leukemia inhibitory <em>factor</em> (LIF), macrophage-colony stimulating <em>factor</em> (M-CSF), monokine inducible by interferon gamma (MIG), macrophage inflammatory protein (MIP)-1 alpha, MIP 2, platelet-derived <em>growth</em> <em>factor</em> BB (PDGF BB), regulated upon activation, normal T-cell expressed, and secreted (Rantes), tumor necrosis <em>factor</em>-alpha (TNF-alpha), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). The proliferation of glial cells as well as the infiltration of inflammatory cells were also evaluated. Although astroglia proliferated from 72 hours post-injury, inflammatory cell dynamics were generally steady. Among cytokines analyzed in the present study, IL-1beta, IL-5, IL-6, IL-12 p40, G-CSF, IFN-gamma, KC, LIF, MIP2, and PDGF BB increased during the early phase of cerebral wound healing, and M-CSF increased during the middle phase, while IL-15, IL-18, and MIG increased during the late phase. In contrast, IL-1alpha, IL-10, IL-12 p70, and TNF-alpha were suppressed throughout the cerebral wound healing process. Based on our findings, quantitative cytokine analyses at the cerebral wound site may be a useful tool for wound age estimation. Further, this study suggests that multiplex data gained from the same sample using a single methodology demonstrates highly accurate cytokine interactions during the process of cerebral wound healing.

Endothelial cells from autopsy and biopsy specimens from a variety of adult human vascular tissue were harvested by collagenase treatment and gentle swabbing of the lumenal surface. Nutrient medium MCDB 107 containing a partially purified brain-derived <em>growth</em> <em>factor</em> (5 micrograms/ml), epidermal <em>growth</em> <em>factor</em> (10 ng/ml) and only 2% (v/v) fetal bovine serum supported clonal and long-term serial culture (<em>17</em>.6 to 26.1 cumulative population doublings) of endothelial cells from vena cava, thoracic aorta and tibial arteries at a 70% rate of success. Cumulative doublings of the cell population from eight cultures were inversely proportional to age of donor of the vascular tissue from which cells were isolated. Heparin had an enhancing effect on cell <em>growth</em> that varied with cell strain. Prostacyclin production of human adult endothelial cell cultures was stimulated by arachidonate and thrombin by <em>17</em> to 20 and 2 to 3-fold respectively. Endogenous and stimulated rates of prostacyclin production by human adult endothelial cells were 2 to 3 times that of human adult smooth muscle cells and 20 to 30 times that of human <em>fibroblasts</em>.

Gab1 is a scaffolding/docking protein that has been suggested to play a role in signal transduction downstream of certain plasma membrane receptors, including platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptors. We found that PDGF induced a rapid Gab1 phosphorylation, which depended on the recruitment of Grb2, indicating that Grb2 acts as a bridge between Gab1 and the PDGF beta-receptor. PDGF also enhanced the binding of Gab1 to the phosphatase SHP-2, but not to p85. To further study the role of Gab1 in PDGF signaling, we transfected porcine aortic endothelial cells with a doxycycline-inducible Gab1 construct. Increased Gab1 expression enhanced the recruitment and activation of SHP-2, as well as the phosphorylation of the mitogen-activated protein kinases Erk and p38 by PDGF. Gab1 expression also enhanced the formation of lamellipodia and cellular protrusions. In Gab1-deficient mouse embryonic <em>fibroblasts</em>, the same phenotype was induced by restoring the expression of wild-type Gab1, but not a mutant Gab1 that was unable to associate with SHP-2. These effects of PDGF on the actin cytoskeleton were not altered by the inhibition of p38 or Erk, but could be blocked by a dominant-negative form of Rac (Asn(<em>17</em>)). Finally, Gab1-deficient <em>fibroblasts</em> showed a decreased chemotactic response toward gradients of PDGF as compared with wild-type cells. In conclusion, Gab1 plays a selective role in the regulation of the mitogen-activated protein kinases Erk and p38 downstream of the PDGF beta-receptor, and contributes to cytoskeletal reorganization and chemotaxis in response to PDGF.

BACKGROUND

Reduction of intramedullary hematopoiesis and the development of myelofibrosis and splenic hematopoiesis are frequent complications of clonal myeloid disorders that cause severe morbidity and death and present a therapeutic challenge. However, the pathogenesis of these complications is still unknown. We evaluated the effect of fibroblast growth factor 2 (FGF-2), the level of which is elevated in patients with clonal myeloid disorders, on bone marrow stromal cell expression of stromal cell-derived factor 1 (SDF-1), a chemokine that is essential for normal hematopoiesis.

METHODS

Reverse transcription-polymerase chain reaction analysis, immunoblot analysis, and enzyme-linked immunosorbent assays were used to examine effects of human recombinant FGF-2 exposure on SDF-1 expression in mouse stromal MS-5 and S-17 cells. Cocultures of human CD34-positive peripheral blood stem cells or mouse pre-B DW34 cells with mouse stromal cells were used to characterize the functional relevance of the effects of FGF-2 on SDF-1 expression. The in vivo hematologic effects of FGF-2 were determined by systemic administration to mice (n = 10). All statistical tests were two-sided.

RESULTS

FGF-2 reduced constitutive SDF-1 mRNA expression and secretion in stromal cells (SDF-1 levels in supernatants: MS-5 cells cultured for 3 days in medium only versus in medium with FGF-2, 95.4 ng/mL versus 22.2 ng/mL, difference = 73.2 ng/mL, 95% confidence interval [CI] = 60.52 to 85.87 ng/mL; P = .002, two-sided Student's t test; S-17 cultured in medium only versus in medium with FGF-2, 203.53 ng/mL versus 32.36 ng/mL, difference = 171.17 ng/mL, 95% CI = 161.8 to 180.6 ng/mL; P<.001). These effects of FGF-2 were reversible. FGF-2 compromised stromal cell support of the growth and survival of pre-B DW34 and myeloid lineage cells, and these effects were reversed in part by exogenous recombinant SDF-1alpha (rSDF-1alpha) (DW34 pre-B cells recovery on S-17 stromal cells, expressed as a percentage of DW34 cells recovered from medium only: with FGF-2 versus without FGF-2, 27.6% versus 100%, difference = 72.4%, 95% CI = 45.34% to 99.51%, P = .008; with FGF-2 plus rSDF1 versus with FGF-2 only, 60.3% versus 27.6%, difference = 32.7%, 95% CI = 9.35% to 56.08%, P = .034; fold increase in number of myeloid lineage cells after culture on S-17 stromal cells: with FGF-2 versus without FGF-2, 0.25-fold versus 3.8-fold, difference = 3.55-fold, 95% CI = 2.66- to 4.44-fold, P<.001; recovery of myeloid cells on S-17 stromal cells, expressed as a percentage of myeloid cells recovered from medium only: FGF-2 plus rSDF-1alpha versus FGF-2 only, 76.5% versus 32.4%, difference = 44.1%, 95% CI = 32.58% to 55.68%, P<.001). Administration of FGF-2 to mice reversibly reduced bone marrow levels of SDF-1 and cellularity and induced immature myeloid cell mobilization, extramedullary hematopoiesis, and splenomegaly.

CONCLUSIONS

Systemic administration of FGF-2 in mice disrupts normal bone marrow hematopoiesis in part through reduced expression of SDF-1. Thus, endogenous FGF-2 may represent a potential therapeutic target in clonal myeloid disorders characterized by bone marrow failure.

OBJECTIVE

The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity.

METHODS

Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor-2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol-3-phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages.

RESULTS

The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 muM insulin, resulted in lower doubling times at all seeding densities tested (0.05 x 10(4) to 1.5 x 10(4)) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol-3-phosphate dehydrogenase activity, 493 +/- 215 vs. 41 +/- 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium.

CONCLUSIONS

The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.

The activity of <em>growth</em> <em>factors</em> is crucial for tumor progression. We previously characterized a secreted <em>fibroblast</em> <em>growth</em> <em>factor</em>-binding protein (FGF-BP1) as a chaperone molecule, which enhances the biological functions of FGFs by releasing FGFs from the extracellular matrix. Here, we characterize the frequency and pattern of FGF-BP1 expression during the malignant progression of pancreas and colorectal carcinoma. For this, we generated monoclonal antibodies that detect FGF-BP1 protein in formalin-fixed, paraffin-embedded tissues and applied in situ hybridization to detect FGF-BP1 mRNA in adjacent tissue sections. FGF-BP1 protein and mRNA were found up-regulated (>70% positive) in parallel (r = 0.70, P < 0.0001) in colon adenoma (n = 9) as well as primary (n = 46) and metastatic (n = 71) colorectal cancers relative to normal colon epithelia (all P < 0.0001, versus normal). Similarly, pancreatitis (n = <em>17</em>), pancreatic intraepithelial neoplasia (n = 80), and pancreatic adenocarcinoma (n = 67) showed a significant up-regulation of FGF-BP1 compared with normal pancreas (n = 42; all P < 0.0001, relative to normal). Furthermore, the biological activity of FGF-BP1 is neutralized by one of the antibodies, suggesting the potential for antibody-based therapeutic targeting. We propose that the up-regulation of the secreted FGF-BP1 protein during initiation of pancreas and colon neoplasia could make this protein a possible serum marker indicating the presence of high-risk premalignant lesions.

OBJECTIVE

The study aimed to verify the safety and feasibility of applying acidic fibroblast growth factor (aFGF) with fibrin glue in combination with surgical neurolysis for nonacute spinal cord injury.

METHODS

This open-label, prospective, uncontrolled human clinical trial recruited 60 patients with spinal cord injuries (30 cervical and 30 thoracolumbar). The mean patient age was 36.5 ± 15.33 (mean ± SD) years, and the male/female ratio was 3:1. The mean time from injury to treatment was 25.7 ± 26.58 months, and the cause of injury included motor vehicle accident (26 patients [43.3%]), fall from a height (17 patients [28.3%]), sports (4 patients [6.7%]), and other (13 patients [21.7%]). Application of aFGF with fibrin glue and duraplasty was performed via laminectomy, and an adjuvant booster of combined aFGF and fibrin glue (2 ml) was given at 3 and 6 months postsurgery via lumbar puncture. Outcome measurements included the American Spinal Injury Association (ASIA) motor scores, sensory scores, impairment scales, and neurological levels. Examination of functional independence measures, visual analog scale, MR imaging, electrophysiological and urodynamic studies, hematology and biochemistry tests, tumor markers, and serum inflammatory cytokines were all conducted. All adverse events were monitored and reported. Exclusions were based on refusal, unrelated adverse events, or failure to participate in the planned rehabilitation.

RESULTS

Forty-nine patients (26 with cervical and 23 with thoracolumbar injuries) completed the 24-month trial. Compared with preoperative conditions, the 24-month postoperative ASIA motor scores improved significantly in the cervical group (from 27.6 ± 15.55 to 37.0 ± 19.93, p < 0.001) and thoracolumbar group (from 56.8 ± 9.21 to 60.7 ± 10.10, p < 0.001). The ASIA sensory scores also demonstrated significant improvement in light touch and pinprick in both groups: from 55.8 ± 24.89 to 59.8 ± 26.47 (p = 0.049) and 56.3 ± 23.36 to 62.3 ± 24.87 (p = 0.003), respectively, in the cervical group and from 75.7 ± 15.65 to 79.2 ± 15.81 (p < 0.001) and 78.2 ± 14.72 to 82.7 ± 16.60 (p < 0.001), respectively, in the thoracolumbar group. At 24-month follow-up, the ASIA impairment scale improved significantly in both groups (30% cervical [p = 0.011] and 30% thoracolumbar [p = 0.003]). There was also significant improvement in neurological level in the cervical (from 5.17 ± 1.60 to 6.27 ± 3.27, p = 0.022) and thoracolumbar (from 18.03 ± 4.19 to 18.67 ± 3.96, p = 0.001) groups. The average sum of motor items in functional independence measure also had significant improvement in both groups (p < 0.05). The walking/wheelchair locomotion subscale showed increased percentages of patients who were ambulatory (from 3.4% to 13.8% and from 17.9% to 35.7% in the cervical and thoracolumbar groups, respectively). There were no related adverse events.

CONCLUSIONS

The use of aFGF for spinal cord injury was safe and feasible in the present trial. There were significant improvements in ASIA motor and sensory scale scores, ASIA impairment scales, neurological levels, and functional independence measure at 24 months after treatment. Further large-scale, randomized, and controlled investigations are warranted to evaluate the efficacy and long-term results.

Purpose: Cholangiocarcinoma (CCA) is a desmoplastic tumor of the biliary tree in which epidermal <em>growth</em> <em>factor</em> receptor (EGFR) is overexpressed and contributes to cancer progression. Although EGFR has been envisaged as a target for therapy, treatment with tyrosine kinase inhibitors (TKI) such as erlotinib did not provide therapeutic benefit in patients with CCA, emphasizing the need to investigate resistance mechanisms against EGFR inhibition.Experimental Design: Resistant CCA cells to EGFR inhibition were obtained upon long-time exposure of cells with erlotinib. Cell signaling, viability, migration, and spheroid <em>growth</em> were determined in vitro, and tumor <em>growth</em> was evaluated in CCA xenograft models.Results: Erlotinib-resistant CCA cells displayed metastasis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial-mesenchymal transition (EMT) and a cancer stem cell (CSC)-like phenotype. Resistant cells exhibited an upregulation of insulin receptor (IR) and insulin-like <em>growth</em> <em>factor</em> (IGF) 1 receptor (IGF1R), along with an increase in IGF2 expression. IR/IGF1R inhibition reduced EMT and CSC-like traits in resistant cells. In vivo, tumors developed from resistant CCA cells were larger and exhibited a more prominent stromal compartment, enriched in cancer-associated <em>fibroblasts</em> (CAF). Pharmacological coinhibition of EGFR and IR/IGF1R reduced tumor <em>growth</em> and stromal compartment in resistant tumors. Modeling of CCA-CAF crosstalk showed that IGF2 expressed by <em>fibroblasts</em> boosted IR/IGF1R signaling in resistant cells. Furthermore, IR/IGF1R signaling positively regulated <em>fibroblast</em> proliferation and activation.Conclusions: To escape EGFR-TKI treatment, CCA tumor cells develop an adaptive mechanism by undergoing an IR/IGF1R-dependent phenotypic switch, involving a contribution of stromal cells. Clin Cancer Res; 24(<em>17</em>); 4282-96. ©2018 AACR.

Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether <em>fibroblasts</em> can substitute <em>growth</em> <em>factors</em> needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different <em>fibroblast</em> numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of <em>fibroblasts</em> the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of <em>fibroblasts</em> keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of <em>fibroblasts</em> showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of <em>fibroblasts</em> present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of <em>fibroblasts</em>. Keratin 6 was downregulated with increasing numbers of <em>fibroblasts</em>, and keratins 16 and <em>17</em> were absent in <em>fibroblast</em>-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of <em>fibroblasts</em> incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In <em>fibroblast</em>-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte <em>growth</em> <em>factor</em>. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with <em>fibroblasts</em>.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of <em>17</em> Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.

Using immunohistochemical techniques a subpopulation of endocrine cells in the human oxyntic mucosa was found to react with antibodies against basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). These cells were identified as histamine-producing enterochromaffin-like (ECL) cells and, to a minor extent, serotonin-producing enterochromaffin cells. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> immunoreactive cells were most frequently found in hyperplastic lesions of ECL cells occurring in hypergastrinemic patients (20 of 27 cases) and in ECL cell carcinoid tumors (10 of <em>17</em> cases). In addition, bFGF mRNA was demonstrated by Northern blot analysis of homogenates from two gastric carcinoids cytologically characterized as pure ECL cell tumors. Although the function of bFGF in normal cells remains unknown, its production in neoplastic conditions may be responsible for the associated desmoplastic and angioblastic proliferations. Moreover, secretion of bFGF by hyperplastic or neoplastic ECL cells may contribute to the circulating levels of the bFGF-like mitogenic <em>factor</em> identified in patients affected by multiple endocrine neoplasia type 1 syndrome.

The role of endogenous <em>growth</em> <em>factors</em> in normal wound healing is not clear. Most of the data on <em>growth</em> <em>factors</em> in healing wounds have been obtained from the application of recombinant exogenous <em>growth</em> <em>factors</em> to animal and human wounds. We describe the immunolocalization of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in the injured dermis of skin from patients with partial and full-thickness burns. Three antibodies demonstrate an extracellular staining pattern of bFGF corresponding to areas of tissue injury that was most intense in specimens collected between 4 and 11 days post-burn injury. In contrast, bFGF staining appeared markedly decreased by Postburn Day <em>17</em> and was more consistent with uninjured tissue in a 30-day-old burn that had virtually reepithelialized. Basic FGF staining in the non-burned skin from the same patients was restricted to the dermal capillary basement membranes and the sweat glands, which is consistent with other reports of immunoreactive bFGF localization in normal adult skin. The immunohistochemical results were confirmed with Western immunoblots of the same tissue. The major band at 16.5 kDa, which is within the recognized range of the bFGF molecule's several forms, was detected in both burned and unburned tissue from the same patient. These findings support the hypothesis that bFGF is a presynthesized mediator that is stored in either the cells or extracellular matrix, is released locally from sites of direct injury, and may be important in early wound healing.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR) play a major role in angiogenesis and are desirable targets for the development of therapeutics. Groups of Wistar Han rats were dosed orally once daily for 4 days with a small molecule pan-FGFR inhibitor (5mg/kg) or once daily for 6 days with a small molecule MEK inhibitor (3mg/kg). Serum phosphorous and FGF23 levels increased in all rats during the course of the study. Histologically, rats dosed with either drug exhibited multifocal, multiorgan soft tissue mineralization. Expression levels of the sodium phosphate transporter Npt2a and the vitamin D-metabolizing enzymes Cyp24a1 and Cyp27b1 were modulated in kidneys of animals dosed with the pan-FGFR inhibitor. Both inhibitors decreased ERK phosphorylation in the kidneys and inhibited FGF23-induced ERK phosphorylation in vitro in a dose-dependent manner. A separate cardiovascular outcome study was performed to monitor hemodynamics and cardiac structure and function of telemetered rats dosed with either the pan-FGFR inhibitor or MEK inhibitor for 3 days. Both compounds increased blood pressure (~+ <em>17</em> mmHg), decreased heart rate (~-75 bpm), and modulated echocardiography parameters. Our data suggest that inhibition of FGFR signaling following administration of either pan-FGFR inhibitor or MEK inhibitor interferes with the FGF23 pathway, predisposing animals to hyperphosphatemia and a tumoral calcinosis-like syndrome in rodents.

OBJECTIVE

To analyze intraocular growth factor and cytokine concentrations in eyes with different stages of age-related macular degeneration (AMD) compared with controls.

METHODS

The Clinical Age-Related Maculopathy Staging (CARMS) system was used for assignment of patients into the respective categories. Aqueous humor specimens were taken before cataract surgery in 21 controls (CARMS 1) and in 17 early (CARMS 2) and 16 intermediate (CARMS 3) AMD patients. In 18 neovascular (CARMS 5) AMD patients, specimens were taken immediately before anti-vascular endothelial growth factor intravitreal therapy. Luminex multiplex bead assays were conducted for endostatin, angiogenin, vascular endothelial growth factor, platelet-derived growth factor AA, placental growth factor, thrombospondin 2, and fibroblast growth factor a.

RESULTS

Vascular endothelial growth factor concentrations were elevated in CARMS 3 (P = 0.037) and tended to be elevated in CARMS 5 (P = 0.093), whereas levels in CARMS 2 (P = 0.425) were similar to CARMS 1. Platelet-derived growth factor levels were diminished in CARMS 2 (P = 0.020), with a trend to lower levels for CARMS 3 (P = 0.099) and CARMS 5 (P = 0.082) compared with CARMS 1. For CARMS 5, antiangiogenic endostatin was elevated (P < 0.002), while antiangiogenic thrombospondin 2 was reduced (P = 0.029).

CONCLUSIONS

Clinical Age-Related Maculopathy Staging 3 dry AMD was associated with higher vascular endothelial growth factor levels than CARMS 5 neovascular AMD. Therefore, intraocular vascular endothelial growth factor concentrations do not seem to reflect choroidal neovascularization activity in neovascular AMD directly. Platelet-derived growth factor was decreased in most forms of AMD. The antiangiogenic endostatin was exclusively elevated in neovascular AMD, while thrombospondin 2 was reduced. Age-related macular degeneration disease seems to be associated with a generally altered cytokine system.

OBJECTIVE

Platelet-rich plasma (PRP) has been increasingly used in sports-related injuries for therapeutic applications. However, there are numerous manufacturing procedures and treatment protocols of PRP use, which make difficult to assess its real efficacy for tissue healing. This study addressed to evaluate the therapeutic effects of locally delivered plasma rich in growth factors (PRGF-Endoret) on the early healing of medial collateral ligament (MCL) in rabbit knees.

METHODS

Thirty-one Japanese white rabbits were subjected to a mop-end tear in the MCL of the left knee. PRGF-Endoret was prepared using Anitua's technique. Two groups were set up. In 17 knees, prepared 1.0 ml of PRGF-Endoret after clotting was applied on the tear site, while in 14 knees the tear site was untreated serving as a control. Quantitative aspects of PRGF-Endoret, the concentration of platelets, leukocytes and erythrocytes and therapeutic growth factors such as PDGF-BB and TGF-β1 were measured. Rabbits were sacrificed at 3 and 6 weeks after the operation and histological and biomechanical evaluation were performed.

RESULTS

No leukocytes were measured and certain amount of growth factors such as PDGF-BB and TGF-β1 were confirmed in the PRGF-Endoret. PRGF-Endoret stimulated proliferation of fibroblasts and neovascularization, and induced statistically better structural properties in repaired MCL.

CONCLUSIONS

Our findings provide evidence that local administration of PRGF-Endoret promotes early steps in ligament healing and the repair of structural properties in a rabbit model. PRGF-Endoret would be a useful product in clinical treatment of ligament injuries.

BACKGROUND

We hypothesized that immune mediator concentrations in the bronchoalveolar fluid (BALF) are predictive of bronchiolitis obliterans syndrome (BOS) and demonstrate specific patterns of dysregulation, depending on the presence of acute cellular rejection, BOS, aspiration, and timing of lung transplantation.

METHODS

We prospectively collected 257 BALF samples from 105 lung transplant recipients. The BALF samples were assessed for absolute and differential white blood cell counts and 34 proteins implicated in pulmonary immunity, inflammation, fibrosis, and aspiration.

RESULTS

There were elevated BALF concentrations of interleukin (IL)-15, IL-<em>17</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, tumor necrosis <em>factor</em>-α, and myeloperoxidase, and reduced concentrations of α1-antitrypsin, which were predictive of early-onset BOS. Patients with BOS had an increased percentage of BALF lymphocytes and neutrophils, with a reduced percentage of macrophages (p < 0.05). The BALF concentrations of IL-1β; IL-8; interferon-γ-induced protein 10; regulated upon activation, normal T-cell expressed and secreted; neutrophil elastase; and pepsin were higher in patients with BOS (p < 0.05). Among those with BOS, BALF concentrations of IL-1RA; IL-8; eotaxin; interferon-γ-induced protein 10; regulated upon activation, normal T-cell expressed and secreted; myeloperoxidase; and neutrophil elastase were positively correlated with time since transplantation (p < 0.01). Those with worse grades of acute cellular rejection had an increased percentage of lymphocytes in their BALF (p < 0.0001) and reduced BALF concentrations of IL-1β, IL-7, IL-9, IL-12, granulocyte colony-stimulating <em>factor</em>, granulocyte-macrophage colony-stimulating <em>factor</em>, interferon-γ, and vascular endothelial <em>growth</em> <em>factor</em> (p ≤ 0.001). Patients with aspiration based on detectable pepsin had increased percentage of neutrophils (p < 0.001) and reduced BALF concentrations of IL-12 (p < 0.001).

CONCLUSIONS

The BALF levels of IL-15, IL-<em>17</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, tumor necrosis <em>factor</em>-α, myeloperoxidase, and α1-antitrypsin at 6 to 12 months after lung transplantation are predictive of early-onset BOS, and those with BOS and aspiration have an augmented chemotactic and inflammatory balance of pulmonary leukocytes and immune mediators. These data justify the surgical prevention of aspiration and argue for the refinement of antirejection regimens.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> play critical roles in many aspects of embryo patterning that are conserved across broad phylogenetic distances. To help understand the evolution of <em>fibroblast</em> <em>growth</em> <em>factor</em> functions, we identified members of the Fgf8/<em>17</em>/18-subfamily in the three-spine stickleback Gasterosteus aculeatus, and investigated their evolutionary relationships and expression patterns. We found that fgf<em>17</em>b is the ortholog of tetrapod Fgf<em>17</em>, whereas the teleost genes called fgf8 and fgf<em>17</em>a are duplicates of the tetrapod gene Fgf8, and thus should be called fgf8a and fgf8b. Phylogenetic analysis supports the view that the Fgf8/<em>17</em>/18-subfamily expanded during the ray-fin fish genome duplication. In situ hybridization experiments showed that stickleback fgf8 duplicates exhibited common and unique expression patterns, indicating that tissue specialization followed the gene duplication event. Moreover, direct comparison of stickleback and zebrafish embryonic expression patterns of fgf8 co-orthologs suggested lineage-specific independent subfunction partitioning and the acquisition or the loss of ortholog functions. In tetrapods, Fgf8 plays an important role in the apical ectodermal ridge of the developing pectoral appendage. Surprisingly, differences in the expression of fgf8a in the apical ectodermal ridge of the pectoral fin bud in zebrafish and stickleback, coupled with the role of fgf16 and fgf24 in teleost pectoral appendage show that different Fgf genes may play similar roles in limb development in various vertebrates.

OBJECTIVE

The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening.

METHODS

The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting.

RESULTS

Pretreatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%.

CONCLUSIONS

Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure.

We have previously reported that the partial estrogen antagonists, tamoxifen, clomiphene and nafoxidine, inhibited angiogenesis in vivo in a dose-related manner in the six-day old chick egg chorioallantoic membrane (CAM) assay. In the present study, we investigated the effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) on the <em>growth</em> of porcine pulmonary artery and human dermal microvascular endothelial cells. Both of these <em>growth</em> <em>factors</em> significantly increased the <em>growth</em> of these cells. The antiproliferative activity of the partial antiestrogens, tamoxifen, nafoxidine and clomiphene, and the pure antiestrogen, ICI 182,780, was determined. Tamoxifen, clomiphene, nafoxidine and ICI 182,780 significantly inhibited endothelial cell <em>growth</em> stimulated by bFGF and VEGF. This inhibition of endothelial cells was not altered by the presence of up to 30 microM of estradiol-<em>17</em> beta. These results indicate that the antiangiogenic action of the antiestrogens does not occur via the estrogen receptor, but by a direct inhibition of <em>growth</em> <em>factor</em> stimulated endothelial cell <em>growth</em>.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy associated with psoriasis and included in seronegative spondyloarthropathy. PsA has several unique characteristics different from rheumatoid arthritis (RA), such as enthesopathy, dactylitis, and abnormal bone remodeling. As compared with synovitis of RA (pannus), proliferation of PsA synovium is mild and characterized by hypervascularity and increased infiltration of polymorphonuclear leukocytes in the synovial tissues. Angiogenesis plays a crucial role in cutaneous psoriasis, and several angiogenic <em>factors</em> such as vascular endothelial <em>growth</em> <em>factor</em>, interleukin-8, angiopoietin, tumor necrosis <em>factor</em>- α and transforming <em>growth</em> <em>factor</em>-β, are suggested to play an important role also in the pathophysiology of PsA. Further, IL-<em>17</em> has various functions such as upregulation of proinflammatory cytokines, attraction of neutrophils, stimulation of keratinocytes, endothelial cell migration, and osteoclast formation via RANKL from activated synovial <em>fibroblasts</em>. Thus, IL-<em>17</em> may be important in angiogenesis, fibrogenesis, and osteoclastogenesis in PsA. In this paper, roles of angiogenesis in the psoriatic synovium are discussed, which may strengthen the understanding of the pathogenesis of PsA.