BACKGROUND

Diabetic foot ulcers (DFUs) represent a significant source of morbidity and an enormous financial burden. Standard care for DFUs involves systemic glucose control, ensuring adequate perfusion, debridement of nonviable tissue, off-loading, control of infection, local wound care and patient education, all administered by a multidisciplinary team. Unfortunately, even with the best standard of care (SOC) available, only 24% or 30% of DFUs will heal at weeks 12 or <em>20</em>, respectively.The extracellular matrix (ECM) in DFUs is abnormal and its impairment has been proposed as a key target for new therapeutic devices. These devices intend to replace the aberrant ECM by implanting a matrix, either devoid of cells or enhanced with <em>fibroblasts</em>, keratinocytes or both as well as various <em>growth</em> <em>factors</em>. These new bioengineered skin substitutes are proposed to encourage angiogenesis and in-<em>growth</em> of new tissue, and to utilize living cells to generate cytokines needed for wound repair.To date, the efficacy of bioengineered ECM containing live cellular elements for improving healing above that of a SOC control group has not been compared with the efficacy of an ECM devoid of cells relative to the same SOC. Our hypothesis is that there is no difference in the improved healing effected by either of these two product types relative to SOC.

METHODS

To test this hypothesis we propose a randomized, single-blind, clinical trial with three arms: SOC, SOC plus Dermagraft® (bioengineered ECM containing living fibroblasts) and SOC plus Oasis® (ECM devoid of living cells) in patients with nonhealing DFUs. The primary outcome is the percentage of subjects that achieved complete wound closure by week 12.

CONCLUSIONS

If our hypothesis is correct, then immense cost savings could be realized by using the orders-of-magnitude less expensive acellular ECM device without compromising patient health outcomes. The article describes the protocol proposed to test our hypothesis.

BACKGROUND

Fibrosis is a cardinal feature of small intestinal neuroendocrine tumors (SI-NETs) both in local peritumoral tissue and systemic sites (cardiac). 5-HT, a commonly secreted NET amine, is a known inducer of fibrosis, although the mechanistic basis for it and growth factors regulating fibrosis and proliferation in the tumor microenvironment are unclear. We hypothesized that targeting 5-HT(2B) receptors on tumor cells would inhibit SI-NET 5-HT release and, thereby, fibroblast activation in the tumor microenvironment.

METHODS

We studied the 5-HT(2B) receptor antagonist PRX-08066 in NET cell lines (KRJ-I, H720) and in the coculture system (KRJ-I cells: fibroblastic HEK293 cells) using real time polymerase chain reaction, ELISA, Ki67 immunostaining, and flow cytometry-based caspase 3 assays to assess antiproliferative and profibrotic signaling pathways.

RESULTS

In the 5-HT(2B) expressing SI-NET cell line, KRJ-I, PRX-08066 inhibited proliferation (IC(50) 4.6 x 10(-9)M) and 5-HT secretion (6.9 x 10(-9)M) and decreased ERK1/2 phosphorylation and profibrotic growth factor synthesis and secretion (transforming growth factor beta 1 [TGFbeta1], connective tissue growth factor [CTGF] and fibroblast growth factor [FGF2]). In the KRJ-I:HEK293 coculture system, PRX-08066 significantly decreased 5-HT release (>60%), Ki67 (transcript and immunostaining: 20%-80%), TGFbeta1, CTGF, and FGF2 transcription (20%-50%) in the KRJ-I cell line. 5-HT itself stimulated HEK293 proliferation (25%) and synthesis of TGFbeta1, CTGF and FGF2. PRX-08066 inhibition of KRJ-I function reversed these effects in the coculture system.

CONCLUSIONS

Targeting the 5-HT(2B) receptor may be an effective antiproliferative and antifibrotic strategy for SI-NETs because it inhibits tumor microenvironment fibroblasts as well as NET cells. Fibrosis and proliferation appear to be biologically interfaced neuroendocrine neoplasia domains.

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 17 Kd and a doublet of <em>20</em>-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 (<em>20</em> of 27 cases) and in ECL cell carcinoid tumors (10 of 17 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.

Chronic nonhealing diabetic foot ulcers are a common medical problem that may precede severe complications such as infection, sepsis and limb loss. Current standard methods of treatment are aimed at removing necrotic debris, controlling infection, and relieving chronic pressure on the wound. Unfortunately, healing rates are poor with standard treatment, averaging 12-<em>20</em> weeks in clinical trials. A new strategy for the treatment of diabetic foot ulcers has been developed through tissue engineering, allowing the application of healthy living skin cells to assist in the healing process. It is hoped that the living tissue will release appropriate quantities of <em>growth</em> <em>factors</em>, cytokines and other proteins to stimulate the chronic wound bed and accelerate healing. Dermagraft (Smith & Nephew) is a neonatal-derived bioengineered tissue comprised of dermal <em>fibroblasts</em>. In this article, the structure and behavior of this tissue will be examined, focusing particularly on the randomized clinical trials performed to justify its use in diabetic foot ulcers.

The dose-dependent effect of a 24 h treatment with estradiol (E(2)) (1, 2, 5, 10 nM) and raloxifene (Rx) (1, 5, 10, <em>20</em> microM) on ER alpha and ER beta mRNA expression, collagen bio-synthesis, prolidase activity, MMP-2, MMP-9, insulin-like <em>growth</em> <em>factor</em> I receptor expression (IGF-1R) and beta1-integrin expressions in cultured <em>fibroblasts</em> obtained from postmenopausal women were examined. Both ligands increased mRNA expression of ER compared to control. Rx at 5 and 10 microM concentrations had greater stimulative effect on collagen biosynthesis, prolidase activity and IGF-1R expression compared to E(2) at 2 and 5 nM concentration. Both studied ER ligands had no effect on beta1-integrin receptor expressions. MMP-2 expression was not detected in human skin <em>fibroblast</em> culture. In contrast to estradiol raloxifene inhibited the expression of MMP-9. Raloxifene had stronger positive stimulative effects on collagen biosynthesis, through different biochemical mechanisms, than estradiol in human skin <em>fibroblasts</em> and might reverse some of the postmenopausal changes in skin or connective tissue. Increase of collagen synthesis induced by raloxifene may be activated by both estrogen receptor dependent and independent pathways such as up-regulation of estrogen receptors, up-regulation of IGF receptor, transcriptional regulation of collagen genes by estrogen receptor-raloxifene complex, increasing of prolidase activity or finally by inhibition of MMP-9 expression.

OBJECTIVE

To examine the up-regulation of vascular endothelial growth factor (VEGF) expression by hypoxia, a crucial event leading to neovascularization, as the reduction in VEGF expression may facilitate minimization of adhesion development.

METHODS

Prospective experimental study.

METHODS

University medical center.

METHODS

Five patients with adhesions undergoing laparotomy with excision of adhesions and normal peritoneum.

METHODS

Adhesion and normal peritoneal fibroblasts were treated with dichloroacetic acid (DCA) or NS-398 (a cyclooxygenase-2 [COX-2] inhibitor) for 24 to 48 hours.

METHODS

A real-time reverse transcriptase polymerase chain reaction (RT-PCR) to quantify relative changes in mRNA levels of VEGF from each treatment.

RESULTS

In both normal peritoneal and adhesion fibroblasts, VEGF mRNA was present with statistically significantly higher levels in adhesion fibroblasts (32%). The DCA treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (20%) and normal peritoneal (18%) fibroblasts. The NS-398 treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (25%) and normal peritoneal (16%) fibroblasts.

CONCLUSIONS

Stimulation of aerobic metabolism by DCA or inhibition of COX-2 by NS-398 reduces VEGF expression. Angiogenesis, which is an integral component in the development of dense vascular adhesions, may be reduced by either COX-2 inhibitors or stimulation of aerobic metabolism by DCA.

OBJECTIVE

Clinical use of plasma rich in growth factors requires biochemical product control. We aimed to measure and modulate concentrations of growth factors in solutions deriving from platelet apheresis or whole blood.

METHODS

Growth factor concentrations were measured 5', 10', 20', 30', 60' after CaCl2 was added at 40°C to platelet-apheresis products (n = 39) or after 60' in platelet concentrates from whole blood (n = 13). Growth factor release was also obtained in platelet apheresis a) by incubation at 22°C or 40°C for 10' or 30' (n = 4); b) by repeated freeze-thaw (n = 9).

RESULTS

Fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) isoforms AA and AB and transforming growth factor beta (TGF-β) concentrations (pg/10(9 ) plt) were 25-60% higher in growth factors solutions from whole blood compared to platelet apheresis. Vascular endothelial growth factor (VEGF), TGF-β and PDGF isoforms were released early (5-10') during incubation: TGF-β concentration increased also at 30'. FGF and epidermal growth factor (EGF) were released only after 30'. Incubation at 40°C/10' increased VEGF (+70%) and decreased EGF (-30%) and PDGF-BB (-50%) versus 22°C/30'. Shock significantly increased TGF-β (1.6-fold), EGF (1.5-fold), FGF (4.5-fold) and lowered PDGF isoforms (0.2- to 0.5-fold) versus prolonged incubation at 40°C.

CONCLUSIONS

Platelets from platelet apheresis and whole-blood release all investigated growth factors. The release can be regulated controlling incubation time and/or temperature and performing cell lysis.

BACKGROUND

Apoptosis is a constant feature of reperfusion injury in ischemic cardiac myocytes, leading to late cell death. Since fibroblast growth factors (FGFs) inhibit apoptosis in differentiated cells, we hypothesized that FGF-1 (acidic FGF), in its native form, and a non-mitogenic isoform would attenuate myocardial ischemia-reperfusion- induced apoptosis.

RESULTS

The effect of native and non-mitogenic fibroblast growth factor-1 mutein (FGF-1 and m-FGF-1) on apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method was tested in a rat model of 20 min regional myocardial ischemia and 24h reperfusion. Myocardial ischemia followed by reperfusion resulted in a high myocardial apoptosis rate in the area at risk. When given as a systemic bolus inmediately after myocardial ischemia, both FGF-1 and m-FGF-1 significantly reduced apoptosis (by 60 and 61.2, respectively; p<0.0001).

CONCLUSIONS

The programed myocyte cell death triggered by ischemia-reperfusion injury is attenuated by FGF-1 in its native or non mitogenic isoforms, suggesting that this effect does not depend on the mitogenic properties of this protein. FGF-1 would contribute to the functional preservation of the myocardium after acute myocardial infarction.

OBJECTIVE

Because it is difficult to predict the behavior of thymomas on the basis of morphology alone, other methods for determining tumor aggressiveness must be explored. This study investigated the correlation between angiogenic grade and invasiveness in thymic epithelial tumors.

METHODS

Immunohistochemical studies of 46 surgically resected thymic epithelial tumors (18 noninvasive thymomas, <em>20</em> invasive thymomas, and 8 thymic carcinomas) were conducted. To highlight the microvessels, we used a specific monoclonal antibody against <em>factor</em> VIII. Expression of vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was determined by using polyclonal antibodies.

RESULTS

Mean microvessel density readings for noninvasive thymomas, invasive thymomas, and thymic carcinomas were 4.6 +/- 3.2, 12.4 +/- 7.5, and 34.4 +/- 16.7, respectively. Stages I, II, III, and IV of thymoma had microvessel density readings of 4.6 +/- 3.2, 8.5 +/- 4.3, 13.8 +/- 7.7, and 22.0 +/- 6.8, respectively. These findings suggest a significant correlation between microvessel density and tumor invasiveness. Vascular endothelial <em>growth</em> <em>factor</em> expression in noninvasive thymomas, invasive thymomas, and thymic carcinomas was present in 1 (5.6%) of 18, 11 (55.0%) of <em>20</em>, and 5 (62.5%) of 8 patients, respectively, thereby indicating a distinct association between vascular endothelial <em>growth</em> <em>factor</em> expression and increased microvessel density. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> expression was present in only 8 patients.

CONCLUSIONS

In patients with thymic epithelial tumors, there appears to be a significant correlation between tumor angiogenesis and invasiveness. Furthermore, our data suggests that this angiogenesis in thymic epithelial tumors might be, at least in part, dependent on vascular endothelial growth factor expression.

Elevated plasma fibroblast growth factor 23 (FGF23) is a prognostic marker in chronic kidney disease. Recently, FGF23 was reported to also be a predictive factor in chronic congestive heart failure (HF). To date however, plasma levels in acute decompensated HF (ADHF) have not been reported and myocardial production and distribution of FGF23 in HF is poorly defined. We aimed to determine plasma levels and myocardial production of FGF23 in ADHF.

Plasma FGF23, N-terminal pro B-type natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR) were assessed in 21 ADHF patients and 19 controls. Myocardial gene expression and distribution of FGF23 was determined on left ventricular samples from HF patients and normal controls.

Plasma FGF23 was markedly higher in ADHF patients compared with controls (1498 ± 1238 versus 66 ± 27 RU/mL, P < 0.0001). There were no correlations between FGF23 and eGFR, NT-proBNP, ejection fraction or age. ADHF subjects with eGFR >60 mL/min/1.73 m(2) had FGF23 levels of 1526 ± 1601 RU/mL versus 55 ± 20 RU/mL in controls (P = 0.007). Quantified myocardial FGF23 gene expression was similar between HF patients and controls. Myocardial FGF23 immunostaining was similar between HF patients and controls, with equal distribution throughout cardiomyocytes.

Patients with ADHF had markedly elevated plasma FGF23 levels. Myocardial FGF23 gene expression was present in HF at a similar level as normal controls, and immunohistochemistry showed similar cellular distribution of FGF23 in HF and controls, suggesting that the myocardium does not contribute to the elevated circulating FGF23 in HF.

Circular dorsal ruffles (CDRs) are transient actin-rich ringlike structures that form on the dorsal surface of <em>growth</em>-<em>factor</em> stimulated cells. However, the dynamics and mechanism of formation of CDRs are still unknown. It has been observed that CDR formation leads to stress fibers disappearing near the CDRs. Because stress fiber formation can be modified by substrate stiffness, we examined the effect of substrate stiffness on CDR formation by seeding NIH 3T3 <em>fibroblasts</em> on glass and polydimethylsiloxane substrates of varying stiffnesses from <em>20</em> kPa to 1800 kPa. We found that increasing substrate stiffness increased the lifetime of the CDRs. We developed a mathematical model of the signaling pathways involved in CDR formation to provide insight into this lifetime and size dependence that is linked to substrate stiffness via Rac-Rho antagonism. From the model, increasing stiffness raised mDia1-nucleated stress fiber formation due to Rho activation. The increased stress fibers present increased replenishment of the G-actin pool, therefore prolonging Arp2/3-nucleated CDR formation due to Rac activation. Negative feedback by WAVE-related RacGAP on Rac explained how CDR actin propagates as an excitable wave, much like wave propagation in other excitable medium, e.g., nerve signal transmission.

Brief experimentally induced seizures have been shown to increase the expression of mRNA encoding basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) in specific brain regions. However, the extent to which this change in mRNA affects the expression of FGF-2 protein in these brain regions has not been examined. In the present study, we exposed rats to brief non-injurious seizures to determine whether this treatment would lead to an increase in FGF-2 protein expression in selected brain regions. Because initial results indicated that the elevation of FGF-2 protein was not significant following acute seizure exposure, we examined both acute and chronic seizure treatment to determine whether FGF-2 protein expression could be increased under conditions of repeated seizures. Brief limbic seizures were induced by minimal electroconvulsive shock (ECS) given as daily treatments for 1 (acute) or 7 (chronic) days. FGF-2 protein was measured in hippocampus, rhinal cortex, frontal cortex, and ol<em>factor</em>y bulb at <em>20</em>, 48, and 72 h following the last seizure. No significant increases in FGF-2 protein were observed in any region following acute ECS. In the chronic ECS-treated groups, significantly elevated FGF-2-like immunoreactivity was found in the frontal and rhinal cortex as compared with the same regions from both control and acute ECS animals. Increases after chronic ECS were maximal at <em>20</em> h, and remained significantly elevated as long as 72 h. These increases were predominantly observed for the 24-kDa and 22/22.5-kDa FGF-2 isoforms. Because chronic ECS, which has been shown to be protective against neuronal cell death, induced significantly more FGF-2 immunoreactivity than did acute ECS, we suggest that FGF-2 expression may be an important substrate for the neuroprotective action of non-injurious seizures. A prolonged induction of the high molecular weight isoforms of FGF-2, as occurs after chronic ECS, may selectively reduce the vulnerability of certain brain regions to a variety of neurodegenerative insults.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) has been implicated in a variety of developmental processes including posterior mesoderm and neural patterning. Previous work has led to contradictory roles for FGF in neural induction and anteroposterior neural patterning. Launay et al. (Development 122, 869-880, 1996) suggested a requirement for FGF in anterior neural induction. In contrast, Kroll and Amaya (Development 122, 3173-3183, 1996) and Bang et al. (Development 124, <em>20</em>75-<em>20</em>85, 1997) proposed that FGF is not required for early neural patterning. Here we use a loss-of-function assay to examine whether FGF is required for neural patterning in three experimental situations: (i) in Xenopus early embryos, (ii) in embryonic explants consisting of presumptive dorsal mesoderm and neurectoderm (Keller explants), and (iii) in explants of dorsal ectoderm and posterior mesoderm in which FGF signaling is specifically blocked in the ectoderm. When cultured until tailbud stages, Keller explants develop neural tissue with normal anteroposterior pattern. Overexpression of the dominant-negative FGF receptor (XFD) in Keller explants inhibited the posterior neural markers En-2, Krox-<em>20</em>, and HoxB9, but not the panneural marker nrp-1 and the anterior neurectodermal markers XAG-1 and Xotx-2. Similar results were seen in whole embryos, but only when XFD RNA was targeted to both the dorsal and lateral regions. In contrast, addition of FGF to Keller explants resulted in a shift of the midbrain-hindbrain boundary marker En-2 to a more anterior position normally fated to become cement gland. We also determined whether FGF is required specifically by the neurectoderm for anteroposterior neural patterning. Recombinants of dorsal ectoderm and posterior mesoderm were made in which FGF was specifically blocked in the ectoderm. Spinal cord and hindbrain markers were inhibited in these recombinants, whereas anterior markers and cement gland development were enhanced. Our results demonstrate that FGF is important for posterior development in both mesoderm and neurectoderm and that neural induction and posteriorization represent separable developmental events.

Adult female Fischer 344 (F344) and Sprague-Dawley (SD) rats were treated with estradiol via Silastic implants for 10 and <em>20</em> days. This treatment period in F344 rats is sufficient to produce dramatic hyperplasia of anterior pituitary lactotropes, activation of folliculo-stellate cells (FS) as phagocytes, and reorganization of the blood supply, i.e. hemorrhagic lakes and arteriogenesis from vessels in the adjacent meninges. Estradiol-treated SD rats do not demonstrate a comparable response. We now report intense focal concentrations of cells immunopositive for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) in estradiol-treated F344 rats predominantly near the posterolateral edge of the anterior pituitary, a zone rich in gonadotropes and lactotropes. Immunostaining for FGF, by both light and electron microscopy, revealed that the immunopositive cells were gonadotropes, and that the immunoprecipitate was cytosolic and was most abundant in the cytosol facing the capillaries. Immunostaining for extracellular matrix-associated FGF also revealed foci of positivity at the postero-lateral edge. Estradiol-treated SD rats did not reveal comparable localization for FGF. Morphological analysis and immunolocalization of S-100 protein, a marker for FS cells, revealed that the periphery of the anterior pituitary of estradiol-treated F344 rats included numerous disrupted gonadotropes and, furthermore, was largely devoid of FS cells. This zone was more intact in control F344 rats, but lacked FS cells. The peripheral parenchyma of control and estradiol-treated SD rats was intact compared to that of F344 rats and consistently included FS cells. These results suggest that disruptions of gonadotropes at the pituitary periphery may release FGF, which could then stimulate angiogenesis from blood vessels within the adjacent meninges. The resultant systemic blood supply would stimulate lactotrope hypertrophy and hyperplasia. Since FS cells are known phagocytes within the anterior pituitary, their absence from the periphery of F344 rats may intensify or prolong the effect of the peripherally released FGF.

Prevascularizing an implantable tissue is one strategy to improve oxygen (O(2)) transport throughout larger tissues upon implantation. This study examined the role of hypoxia both during (i.e., as a stimulus) and after (i.e., mimicking implant conditions) vascularization of an implantable tissue. Tissues consisted of microcarrier beads coated with human umbilical vein endothelial cells embedded in fibrin. The fibrin was covered with a monolayer of normal human lung <em>fibroblasts</em> (NHLFs), or exposed to conditioned media from NHLFs. Capillary networks developed at <em>20</em>% or 1% O(2) tension for 8 days. In some experiments, tissues were supplemented with vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, whereas in others the tissues prevascularized at <em>20</em>% O(2) were transferred to 1% O(2) for 8 additional days. Maximal capillary formation occurred in media conditioned by NHLFs at <em>20</em>% O(2), supplemented with VEGF (concentration >10 pM). Hypoxia (1% O(2)) did not stimulate basic <em>fibroblast</em> <em>growth</em> <em>factor</em> production and decreased in vitro angiogenesis, despite an increase in endogenous VEGF production. Hypoxia also degraded a preformed capillary network within 4 days. Hence, strategies to prevascularize implantable tissues may not require the physical presence of stromal cells, but will likely require <em>fibroblast</em>-derived <em>growth</em> <em>factors</em> in addition to VEGF to maintain capillary <em>growth</em>.

After a penetrating lesion in the central nervous system, astrocytes enlarge, divide, and participate in creating an environment that adversely affects neuronal regeneration. We have recently shown that the neural cell adhesion molecule (N-CAM) partially inhibits the division of early postnatal rat astrocytes in vitro. In the present study, we demonstrate that addition of N-CAM, the third immunoglobulin-like domain of N-CAM, or a synthetic decapeptide corresponding to a putative homophilic binding site in N-CAM partially inhibits astrocyte proliferation after a stab lesion in the adult rat brain. Animals were lesioned in the cerebral cortex, hippocampus, or striatum with a Hamilton syringe and needle at defined stereotaxic positions. On one side, the lesions were concomitantly infused with N-CAM or with one of the N-CAM-related molecules. As a control, a peptide of the same composition as the N-CAM decapeptide but of random sequence was infused on the contralateral side of the brain. We consistently found that the population of dividing astrocytes was significantly smaller on the side in which N-CAM or one of the N-CAM-related molecules was infused than on the opposite side. The inhibition was greatest in the cortical lesion sites (approximately 50%) and was less pronounced in the hippocampus (approximately 25%) and striatum (approximately <em>20</em>%). Two weeks after the lesion, the cerebral cortical sites infused with N-CAM continued to exhibit a significantly smaller population of dividing astrocytes than the sites on the opposite side. When N-CAM and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, which is known to stimulate astrocyte division in vitro, were coinfused into cortical lesion sites, astrocyte proliferation was still inhibited. These results suggest the hypothesis that, by reducing glial proliferation, N-CAM or its peptides may help create an environment that is more suitable for neuronal regeneration.

BACKGROUND

Vascular Ehlers-Danlos syndrome (VEDS) causes reduced life expectancy because of arterial dissections/rupture and hollow organ rupture. Although the causative gene, COL3A1, was identified>><em>20</em> years ago, there has been limited progress in understanding the disease mechanisms or identifying treatments.

RESULTS

We studied inflammatory and transforming growth factor-β (TGF-β) signaling biomarkers in plasma and from dermal fibroblasts from patients with VEDS. Analyses were done in terms of clinical disease severity, genotype-phenotype correlations, and body composition and fat deposition alterations. VEDS subjects had increased circulating TGF-β1, TGF-β2, monocyte chemotactic protein-1, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and leptin and decreased interleukin-8 versus controls. VEDS dermal fibroblasts secreted more TGF-β2, whereas downstream canonical/noncanonical TGF-β signaling was not different. Patients with COL3A1 exon skipping mutations had higher plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and VEDS probands had abnormally high plasma C-reactive protein versus affected patients identified through family members before any disease manifestations. Patients with VEDS had higher mean platelet volumes, suggesting increased platelet turnover because of ongoing vascular damage, as well as increased regional truncal adiposity.

CONCLUSIONS

These findings suggest that VEDS is a systemic disease with a major inflammatory component. C-reactive protein is linked to disease state and may be a disease activity marker. No changes in downstream TGF-β signaling and increased platelet turnover suggest that chronic vascular damage may partially explain increased plasma TGF-β1. Finally, we found a novel role for dysregulated TGF-β2, as well as adipocyte dysfunction, as demonstrated through reduced interleukin-8 and elevated leptin in VEDS.

OBJECTIVE

Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly.

METHODS

Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and <em>growth</em> <em>factors</em>. Cells grew out and were subcultured up to <em>20</em> times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy.

RESULTS

Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix.

CONCLUSIONS

These experiments confirmed that MSC cell fate choice depends on minute, cell-derived forces. Applied force could assist in commercial manufacture of cultured bio-engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.

OBJECTIVE

Recent data indicate that oxidized low-density lipoprotein (ox-LDL) has several proatherogenic effects, e.g. induction of macrophage chemoattractants, adhesion molecules, cytokines, type-1 plasminogen activator inhibitor and platelet-derived <em>growth</em> <em>factor</em> A-chain by smooth muscle cells. Therefore, ox-LDL has been utilized as a marker of oxidative modification of proteins in atherosclerosis. Because heart valves consist of smooth muscle cells, <em>fibroblasts</em> and endothelial cells, and because valvular disease and coronary atherosclerosis could result from similar biological processes, we investigated ox-LDL accumulation in isolated aortic and pulmonary valves and coronary arteries from patients with angiographically proven coronary heart disease (CHD, n = 19), patients with idiopathic congestive heart failure (IDCM = idiopathic dilated cardiomyopathy, n = <em>20</em>), and transplant donors.

METHODS

Masson-Goldner staining and immunohistochemistry utilizing anti ox-LDL and CD68 were performed on paraffin sections of freshly isolated semilunar valves. Data were analyzed by digital image planimetry and by visual scoring of staining intensity.

RESULTS

Ox-LDL immunoreactivity was identified in the vascular aspect of the attachment line, in the deep valve stroma, and in the ventricular and vascular endothelium of the semilunar valves, colocalizing with macrophages. Valvular ox-LDL area was significantly increased in CHD-patients (P < 0.03) and IDCM-patients (P < 0.04) compared with controls. More ox-LDL was accumulating in the pulmonary valves than in the aortic valves (P = 0.04) as assessed by area and staining intensity. Valvular ox-LDL area in pulmonary valve and aortic valve was significantly correlated with ox-LDL accumulation in the intimal layer (P < 0.001) and medial layer (P < 0.001) of coronary arteries from the same patients.

CONCLUSIONS

The data suggest that the biological process leading to ox-LDL accumulation in coronary atherosclerosis also involves heart valves. Therefore, accumulation of the oxidative stress marker ox-LDL in heart valves illustrates atherosclerosis as an additional mechanisms accelerating valvular degeneration in these patients.

To investigate the role of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) in chick limb development, we constructed a replication-defective spleen necrosis virus to ectopically express <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 in stage <em>20</em>-22 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, infected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of host wings receiving infected grafts at their anterior borders had duplications of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressively more posterior sites of host limb buds, and grafting to the posterior border had no effect at all. Several techniques were used to determine the role of infected tissue in forming skeletal duplications. First, staining with an <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 specific monoclonal antibody showed higher than endogenous levels of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 expression associated with extra elements. Second, the host/donor composition of duplicated elements was determined by simultaneously infecting donor cells with viruses encoding <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 or beta-galactosidase; donor tissue was then visualized by X-gal staining. Patterns of ectopic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicated structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications caused by infected quail wing bud cells grafted to the chick wing bud. These observations suggest that <em>fibroblast</em> <em>growth</em> <em>factor</em>-2-expressing donor tissue induced host tissue to form normally patterned extra elements. In support of this conclusion, implanting beads containing <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 caused partial duplications of digit 2. These data provide the first direct evidence that <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 plays a role in patterning in the limb bud.

Mineralocorticoid receptors (MRs) are classically known to be expressed in the distal collecting duct of the kidney. Recently it was reported that MR is identified in the heart and vasculature. Although MR expression is also found in the brain, it is restricted to the hippocampus and cerebral cortex under normal condition, and the role played by MRs in brain remodeling after cerebral ischemia remains unclear. In the present study, we used the mouse <em>20</em>-min middle cerebral artery occlusion model to examine the time course of MR expression and activity in the ischemic brain. We found that MR-positive cells remarkably increased in the ischemic striatum, in which MR expression is not observed under normal conditions, during the acute and, especially, subacute phases after stroke and that the majority of MR-expressing cells were astrocytes that migrated to the ischemic core. Treatment with the MR antagonist spironolactone markedly suppressed superoxide production within the infarct area during this period. Quantitative real-time RT-PCR revealed that spironolactone stimulated the expression of neuroprotective or angiogenic <em>factors</em>, such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), whereas immunohistochemical analysis showed astrocytes to be cells expressing bFGF and VEGF. Thereby the incidence of apoptosis was reduced. The up-regulated bFGF and VEGF expression also appeared to promote endogenous angiogenesis and blood flow within the infarct area and to increase the number of neuroblasts migrating toward the ischemic striatum. By these beneficial effects, the infarct volume was significantly reduced in spironolactone-treated mice. Spironolactone may thus provide therapeutic neuroprotective effects in the ischemic brain after stroke.

We have characterized two high affinity acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-<em>20</em> kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-157<em>20</em>). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.

The overexpression of angiogenic <em>factors</em> such as vascular endothelial <em>growth</em> <em>factor</em> (VEGF), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and insulin-like <em>growth</em> <em>factors</em> (IGFs) plays a role in the migration and proliferation of endothelial cells in many cancers. Consequently, we investigated the effects of bombesin/gastrin-releasing peptide (GRP) antagonists on the expression of these angiogenic <em>factors</em>, the activities of matrix metalloproteinases (MMPs)-2 and -9, as well as the vascular density in MDA-MB-435 human oestrogen-independent breast cancers. Nude mice bearing orthotopic xenografts of MDA-MB-435 breast cancers were treated with bombesin/GRP antagonists for 6 weeks. Daily administration of <em>20</em> microg of RC-3095 or 10 microg of RC-3940-II significantly decreased the weight of MDA-MB-435 cancers by 44 and 53%, respectively. The inhibition of tumour <em>growth</em> was associated with a substantial reduction in the expression of mRNA and protein levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), IGF-II and VEGF-A in the tumours. Both bombesin/GRP antagonists significantly decreased the vessel density of the tumours by about 37%, as shown by immunohistochemical detection of vessels on tumour slides. Gelatinolytic activities, detected by zymography, revealed a 33-46% reduction in MMP-9 activity after the treatment with either antagonist. In vitro studies revealed that MDA-MB-435 cells secrete bFGF, IGF-II and VEGF-A, and the secretion of these <em>factors</em> is inhibited by RC-3095 and RC-3940-II. This study demonstrates the antiangiogenic effect of bombesin/GRP antagonists RC-3095 and RC-3940-II, and underscores their possible therapeutic application for treatment of breast cancers.