There is evidence that neuropeptides, especially the opiate receptor agonists, are involved in wound healing. We have previously observed that beta-endorphin, the endogenous ligand for the mu-opiate receptor, stimulates the expression of cytokeratin <em>16</em> in a dose-dependent manner in human skin organ cultures. Cytokeratin <em>16</em> is expressed in hyperproliferative epidermis such as psoriasis and wound healing. Therefore we were interested to study whether epidermal mu-opiate receptor expression is changed at the wound margins in acute and chronic wounds. Using classical and confocal microscopy, we were able to compare the expression level of mu-opiate receptors and the influence of beta-endorphin on transforming <em>growth</em> <em>factor</em> beta type II receptor in organ culture. Our results show indeed a significantly decreased expression of mu-opiate receptors on keratinocytes close to the wound margin of chronic wounds compared to acute wounds. Additionally beta-endorphin upregulates the expression of transforming <em>growth</em> <em>factor</em> beta type II receptor in human skin organ cultures. These results suggest a crucial role of opioid peptides not only in pain control but also in wound healing. Opioid peptides have already been used in animal models in treatment of wounds; they induce <em>fibroblast</em> proliferation and <em>growth</em> of capillaries, and accelerate the maturation of granulation tissue and the epithelization of the defect. Furthermore opioid peptides may fine-tune pain and the inflammatory response while healing takes place. This new knowledge could potentially be used to design new locally applied drugs to improve the healing of painful chronic wounds.

Cyclic stretching and <em>growth</em> <em>factors</em> like TGF-β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal <em>fibroblasts</em>. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-β1 samples. However, at weeks 5 and 7, -TGF-β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-β1 samples. The collagen was better organized in -TGF-β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-β1 samples, which correlated with higher collagen deposition. The TGF-β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were <em>16</em>-fold and 10-fold higher in +TGF-β1 samples, respectively. The role of TGF-β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-β1 signaling during cyclic stretching. One advantage of TGF-β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-β1 to obtain improved tensile properties followed by TGF-β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a highly conserved and ubiquitously distributed mitogen. In amphibian oocytes bFGF mRNA is regulated post-transcriptionally by interaction with an antisense RNA transcript. We used reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridization to determine the presence of bFGF and its antisense RNA (gfg) in unfertilized human oocytes and postnatal differentiated tissues. BFGF and gfg transcripts were co-expressed in many tissues, with bFGF transcripts (7, 3.7 and 1.8 kb) being more abundant than the gfg transcript (1.5 kb) in 8 of <em>16</em> tissues examined. Sense and antisense expression was approximately equal in kidney and colon, while in heart, liver, skeletal muscle and testis gfg transcripts predominated. RT-PCR demonstrated the presence of bFGF and gfg transcripts in unfertilized oocytes where the antisense transcript was present in excess of the sense transcript. These findings suggest a role for gfg in regulation of bFGF expression.

The function of chemokine receptors on structural cells is only partially known. We previously reported the expression of a functional CCR3 receptor on airway epithelial cells (EC). We speculated that CCR3 might drive wound repair and expression of inflammatory genes in epithelium. The human airway EC lines BEAS-2B, <em>16</em>-HBE, and primary bronchial EC were used to test the effect of in vitro challenge with the CCR3 ligands CCL11/eotaxin, CCL24/eotaxin-2, or CCL26/eotaxin-3 on 1) wound repair, using an established wound model; 2) cell proliferation and chemotaxis, using specific fluorometric assays; and 3) gene expression, using pathway-specific arrays for inflammatory and profibrotic cytokines, chemokines, and chemokine receptor genes. Agonist specificity was tested by cell pretreatment with an AstraZeneca CCR3 antagonist (10(-8) - 10(-6) M). CCL24 challenge significantly accelerated epithelial wound closure, with similar effects exerted by CCL11 and CCL26. This effect was time dependent, submaximal at 1 nM, and comparable in potency to epidermal <em>growth</em> <em>factor</em>. CCL24 induced a concentration-dependent increase in EC proliferation and chemotaxis, with significant effects observed at 10 nM. The AstraZeneca compound selectively inhibited these CCL24-mediated responses. CCL11 induced the up-regulation of several profibrogenic molecules such as <em>fibroblast</em> <em>growth</em> <em>factor</em> 1 and 5 and of several CC and CXC chemokines. Epithelial immunostaining for CCR3 was stronger in bronchial biopsies of asthmatics displaying marked inflammatory changes than in nondiseased samples. Epithelial CCR3 participates in key functions for wound repair, amplifies the expression of profibrogenic and chemokine transcripts, and appears up-regulated in inflamed asthmatic airways.

Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) type 1 receptor antagonists have strong cytostatic properties on in vitro cultures of many normal and neoplastic cells. They are effective, in particular, in reducing the <em>growth</em> of human lung <em>fibroblasts</em>, renal canine epithelial cells, bovine adrenal endothelial cells, simian T lymphocytes, and of neoplastic cell lines derived from human neuroblastomas, a ductal pancreatic carcinoma of the Syrian hamsters, human salivary glands adenocarcinomas, and two lines of human breast adenocarcinomas. ACE inhibitors are also effective in protecting lungs, kidneys and bladders from the development of nephropathy, pneumopathy, cystitis, and eventually fibrosis in different models of organ-induced damage such as exposure to radiation, chronic hypoxia, administration of the alkaloid monocrotaline or bladder ligation. ACE inhibitors and AII type 1 receptor antagonists are also effective in reducing excessive vascular neoformation in a model of injury to the cornea of rats and rabbits, and in controlling the excessive angiogenesis observed in the Solt-Farber model of experimentally induced hepatoma, in methylcholantrene or radiation-induced fibrosarcomas, in radiation-induced squamous cell carcinomas and in the MA-<em>16</em> viral-induced mammary carcinoma of the mouse. Captopril was, in addition, effective in controlling tumor <em>growth</em> in a case of Kaposi's sarcoma in humans. The inhibition of AII synthesis and/or its blockade by AII receptors is likely to be an important mechanism for this cytostatic action. The mitogenic effect of AII is well established and a reduction of AII synthesis may well explain cell and neoplasm delayed <em>growth</em>. Moreover, AII regulates and enhances the activity of several <em>growth</em> <em>factors</em> including transforming <em>growth</em> <em>factor</em> B (TGFB) and smooth muscle actin (SMA); and many of these <em>factors</em> are reduced in tissues of animals treated with ACE inhibitors and AII type 1 receptor antagonists. These processes seem to be particularly relevant in the control of <em>fibroblast</em> <em>growth</em> and in the control of the ensuing fibrosis. The ACE inhibitors containing a sulphydril (SH) or thiol radical in their moiety (Captopril and CL242817) seemed to be more effective in controlling fibrosis and the <em>growth</em> of some neoplastic cells than those ACE inhibitors without this thiol radical in their structure, even if the second group of these drugs show in vitro a stronger inhibitory effect on converting enzyme activity. Pharmacologically it is known that ACE inhibitors containing a thiol radical also have antioxidant properties and they are efficient in controlling metalloproteinase action. However, although these additional properties are pharmacologically relevant, the blockade of AII synthesis plays an essential role in the cytostatic activity of these two categories of drugs. These observations underline that in addition to the beneficial effect of these drugs on the cardiovascular system, new potential applications are opening for their wider deployment.

The type beta transforming <em>growth</em> <em>factors</em> (TGF-beta s) are a family of potent cytokines with diverse effects on proliferation, differentiation, turnover of extracellular matrix components, oncogene expression, and other aspects of cellular phenotype. Unlike lung <em>fibroblasts</em> of certain species, unstimulated human lung <em>fibroblast</em> lines produce little or no TGF-beta in culture. However, TGF-beta has been reported to autoregulate its own production in certain human tumor cells and in rodent cell lines. To test whether this phenomenon is operative in <em>fibroblasts</em> from normal human lung tissue, confluent cultures of IMR90 normal fetal lung <em>fibroblasts</em> were exposed to TGF-beta. Cultures were exposed briefly to purified TGF-beta 1 under serum-free conditions and secretion of newly synthesized TGF-beta over the ensuing 72 h was determined by immunoblotting and bioassays made specific with the use of neutralizing antibodies. Steady-state levels of mRNA for TGF-beta 1 were detected by Northern and slot blot hybridization analysis of total cellular RNA. The 2.5 kb TGF-beta 1 mRNA species rose within 1.5 h of exposure of IMR90 cells to TGF-beta 1 and reached maximal levels after <em>16</em> h. Increased levels of TGF-beta were detected in conditioned medium 9 h after the start of the exposure. Thereafter, TGF-beta continued to accumulate at an elevated rate (90 +/- 7 versus < or = 15 pg/10(6) cells/h in uninduced cells) for up to 72 h. As little as 1 ng/ml TGF-beta 1 auto-induced TGF-beta secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived <em>growth</em> <em>factor</em> receptor (PDGFR) β is a marker of stromal pericytes and <em>fibroblasts</em> and represents an interesting target for both diagnosis and therapy of solid tumors. A receptor-specific imaging agent would be a useful tool for further understanding the prognostic role of this receptor in vivo. Affibody molecules constitute a class of very small binding proteins that are highly suited for in vivo imaging applications and that can be selected to specifically recognize a desired target protein. Here we describe the isolation of PDGFRβ-specific Affibody molecules with subnanomolar affinity. First-generation Affibody molecules were generated from a large naive library using phage display selection. Subsequently, sequences from binders having a desired selectivity profile and competing with the natural ligand for binding were used in the design of an affinity maturation library, which was created using a single partially randomized oligonucleotide. From this second-generation library, Affibody molecules with a 10-fold improvement in affinity (K(d)=0.4-0.5 nM) for human PDGFRβ and a 4-fold improvement in affinity (K(d)=6-7 nM) for murine PDGFRβ were isolated and characterized. Complete reversible folding after heating to 90 °C, as demonstrated by circular dichroism analysis, supports tolerance to labeling conditions for molecular imaging. The binders were highly specific, as verified by dot blot showing staining reactivity only with human and murine PDGFRβ, but not with human PDGFRα, or a panel of control proteins including <em>16</em> abundant human serum proteins. The final binder recognized the native conformation of PDGFRβ expressed in murine NIH-3T3 <em>fibroblasts</em> and human AU565 cells, and inhibited ligand-induced receptor phosphorylation in PDGFRβ-transfected porcine aortic endothelial cells. The PDGFRβ-specific Affibody molecule also accumulated around tumoral blood vessels in a model of spontaneous insulinoma, confirming a potential for in vivo targeting.

The interaction between hosts and the viruses that infect them is a dynamic one, and a <em>growing</em> literature documents the fact that many viruses have developed mechanisms designed to avoid elimination by the host immune system. One of the immune strategies used by the host and targeted by virus proteins is apoptosis triggered by the cytokine tumor necrosis <em>factor</em> (TNF). Mouse <em>fibroblast</em> LM cells are spontaneously sensitive to TNF. When the wild-type E6 protein from the human papillomavirus type <em>16</em> (HPV <em>16</em>) was expressed in LM cells, the cells became resistant to TNF. This resistance was examined by several means, including cell morphology, the dose- and time-independent response to TNF in a cell death ELISA, trypan blue exclusion, and cell proliferation. The level of p53 did not rise in TNF-treated cells prior to apoptosis, suggesting a p53-independent mechanism. Significant, though not complete, resistance to TNF was also observed following transfection of a plasmid expressing a mutant E6 protein, which is unable to mediate rapid degradation of the p53 tumor suppressor. These results indicate that the HPV <em>16</em> E6 protein can protect LM cells from TNF-triggered apoptosis and likely does so by a mechanism other than mediation of p53 degradation.

E2F-1 is a member of a family of transcription <em>factors</em> implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type <em>16</em> (HPV<em>16</em>) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited <em>growth</em> of primary and established <em>fibroblasts</em>. The <em>growth</em>-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV<em>16</em> E7 mutants that were unable to cooperate with HPV<em>16</em> E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH-responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased <em>growth</em> plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild-type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from <em>16</em> to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH-mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild-type mice. Adult Prg4 mutant mice had decreased marrow and liver <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) mRNA and reduced serum FGF-2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF-2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone- and liver-derived FGF-2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure.

Insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), a prevalent <em>growth</em> <em>factor</em> secreted by bone cells, has important effects on bone remodeling. Hormones are known to regulate the synthesis of skeletal IGF-I, but there is limited information about the actions of <em>growth</em> <em>factors</em> on IGF-I synthesis. We tested the effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em>-beta 1 (TGF beta 1), and platelet-derived <em>growth</em> <em>factors</em> (PDGF) AA and BB on IGF-I mRNA expression and polypeptide concentrations in cultures of osteoblast-enriched (Ob) cells from 22-day-old fetal rat calvariae. Steady state IGF-I mRNA levels were determined by Northern blot analysis, and IGF-I concentrations were determined in acidified and fractionated culture medium by a specific RIA. Treatment of Ob cells with bFGF at 0.06-6 nM, TGF beta 1 at 0.04-4 nM, and PDGF BB at 0.3-3.3 nM caused a dose-dependent decrease in steady state IGF-I mRNA. A smaller effect was observed with PDGF AA. The effect was initially observed after 6-8 h of treatment and was maximal after <em>16</em> h. Treatment with bFGF at 0.6-6 nM, TGF beta 1 at 0.4-4 nM, and PDGF BB at 0.3-3.3 nM for 24 h decreased IGF-I polypeptide concentrations by 40-80%. The effects of bFGF, TGF beta 1, and PDGF BB and AA on IGF-I mRNA were independent of protein synthesis and cell division, as they were observed in the presence and absence of cycloheximide at 3.6 microM or hydroxyurea at 1 mM. Similarly, their inhibitory actions on immunoreactive IGF-I were not prevented by hydroxyurea. In conclusion, bFGF, TGF beta 1, PDGF BB, and, to a lesser extent, PDGF AA decrease skeletal IGF-I synthesis by reducing IGF-I transcript levels, and this effect may contribute to their actions on selected aspects of Ob cell function.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>16</em> is one of the rare secreted proteins that do not possess a cleavable signal sequence. Here we describe our examination of the mechanism and structural requirements for the secretion of FGF-<em>16</em> from COS-1 transfectants. Inhibition of its secretion by brefeldin A and identification of an N-glycan on the secreted form confirmed that FGF-<em>16</em> is secreted by means of the endoplasmic reticulum and Golgi apparatus, as are secreted proteins having a conventional cleavable signal sequence. Deletion of its N terminus abolished secretion of FGF-<em>16</em>. When chimerized with prolactin, however, the N-terminal sequence of FGF-<em>16</em> was not able to mediate secretion of the chimera. Point mutations that made the N terminus less hydrophobic had little effect on secretion of FGF-<em>16</em>, whereas making the central hydrophobic region less hydrophobic abolished secretion. Within cells, an unsecretable FGF-<em>16</em> N-terminal deletion mutant was distributed in the perinuclear region and overlapped the distribution of the Golgi apparatus. Mutants with less hydrophobic central regions were distributed evenly throughout the cytosol. Collectively, these results indicate that FGF-<em>16</em> employs a unique bipartite signal sequence (i.e. both the N-terminal region and central hydrophobic region) that is not cleaved, although it shares the same secretory machinery used by secreted proteins with cleavable signal sequences.

OBJECTIVE

A hallmark of asthma is airway remodelling, which includes increased deposition of extracellular matrix (ECM) protein. Viral infections may promote the development of asthma and are the most common causes of asthma exacerbations. We evaluated whether rhinovirus (RV) infection induces airway remodelling, as assessed by ECM deposition.

METHODS

Primary human bronchial epithelial cells and lung parenchymal <em>fibroblasts</em> were infected with RV-2 or RV-<em>16</em>, or treated with RV-<em>16</em> RNA, imiquimod (Toll-like receptor (TLR) 7/8 agonist) or polyinosinic : polycytidylic acid (poly I : C) (activator of TLR 3, retinoic-acid-inducible protein I and melanoma-differentiated-associated gene 5). Changes in ECM proteins and their transcription were measured by ELISA and quantitative real-time PCR. In addition, gene expression for ECM proteins was assessed in a mouse model of RV infection.

RESULTS

RV infection increased deposition of the ECM protein, perlecan, by human bronchial epithelial cells, and collagen V and matrix-bound vascular endothelial growth factor were increased in both human bronchial epithelial cell and fibroblast cultures. Purified RV-<em>16</em> RNA, poly I : C and imiquimod induced similar increases in ECM deposition to those observed with RV-infected <em>fibroblasts</em>. However, only poly I : C induced ECM deposition by bronchial epithelial cells, suggesting that RV-induced ECM deposition is mediated through TLR. Furthermore, gene expression for fibronectin and collagen I was increased in lung homogenates of mice infected with RV-1b.

CONCLUSIONS

RV infection and TLR ligands promote ECM deposition in isolated cell systems and RV induces ECM gene expression in vivo, thus demonstrating that RV has the potential to contribute to remodelling of the airways through induction of ECM deposition.

A structural analysis of the rat low-affinity nerve <em>growth</em> <em>factor</em> (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse <em>fibroblasts</em> or COS cells, and the ability of the mutant receptor to bind NGF was assayed. In the first mutant, all but <em>16</em> amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cysteine-rich sequence of the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.

In response to oncogenic insults, normal human cells execute a defense response that culminates in cellular suicide, apoptosis. Normal human diploid <em>fibroblasts</em> expressing the human papillomavirus type <em>16</em> (HPV-<em>16</em>) E7 oncoprotein are predisposed to apoptosis when they are deprived of <em>growth</em> <em>factors</em>. Even though a dominant negative p53 mutant abrogates the cell death response, it is not accompanied by p53 phosphorylation, the DNA binding capacity of p53 remains unaltered, and no activation of common p53-dependent transcriptional targets is observed. Expression of two insulin-like <em>growth</em> <em>factor</em>-1 binding proteins, IGFBP-2 and -5, is increased presumably in response to enhanced NF-kappaB activity in HPV-<em>16</em> E7-expressing serum-starved cells. Phosphorylation of AKT, an important modulator of IGF-1 survival signaling, is lower in serum-starved E7-expressing cells, and exogenously added IGF-1 can partially inhibit the cell death response. This suggests that IGFBP-2 and -5 may limit IGF-1 availability thus decreasing survival signaling. Caspase 3 but not caspase 8 is activated in serum-starved HPV-<em>16</em> E7-expressing cells. Caspase inhibition affects nuclear DNA fragmentation, but cell death is not inhibited. Although mitochondria play important roles in caspase-dependent as well as -independent forms of cell death, there is no evidence for cytochrome c release and thus for mitochondrial permeabilization in <em>growth</em> <em>factor</em> deprived HPV-<em>16</em> E7-expressing cells.

While the field of tissue engineering is a burgeoning one, progress with fat engineering has lagged, due in large part to problems associated with nurturing and sustaining this unique tissue in vivo. In a pilot study using an experimental rat model, we induced liponeogenesis with a combination of Matrigel and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in an fibrovascular scaffold, isolating the construct to a pedicled blood supply (the superficial inferior epigastric vessels) via a silicone housing, creating an engineered three-dimensional adipose tissue construct. Adipose tissue and vascular in<em>growth</em> were assessed histologically and followed by serial study at 4-week intervals for <em>16</em> weeks. We demonstrated persistence of shaped adipose tissue constructs over time, and postulate that incorporating a vascular supply may enhance the durability of experimentally induced fat constructs and potentially provide a means for microsurgical transfer of the construct.

Penetration of basement membrane layers is a hallmark feature of metastatic tumor cells. The invasive propensity of murine SCC-VII squamous carcinoma cells is in part attributable to the extracellular action of the lysosomal cysteine proteinase cathepsin B. Although most noncancerous cells store this enzyme in the lysosomes, we found that SCC-VII cells release a large fraction (42%) of their newly synthesized procathepsin B into the culture medium. Procathepsins D and L, the precursors of other major lysosomal proteinases, are also secreted in significant amounts (24 and 75%, respectively). In contrast, normal murine 3T3-L1 <em>fibroblasts</em> exocytose only minor amounts of their newly synthesized procathepsins B (10%), D (<1%), and L (<em>16</em>%). Western blotting analysis revealed that SCC-VII cells are deficient in the 300 kDa mannose 6-phosphate/insulin-like <em>growth</em> <em>factor</em>-II receptor (M6P/IGF2R), a tumor suppressor with a central role in the intracellular transport of lysosomal enzymes. Consistent with the absence of M6P/IGF2R, SCC-VII cells lack dense lysosomes, with the bulk of intracellular acid hydrolases residing in late endosomes/ prelysosomes. On the other hand, the synthesis of the M6P recognition marker on lysosomal enzymes is not impaired in SCC-VII cells, because [33P]Pi is incorporated into the carbohydrate moieties of procathepsins B, D, and L. Furthermore, 69% of the phosphorylated N-linked oligosaccharides synthesized by SCC-VII cells carry phosphomonoester groups and as such constitute high-affinity ligands for M6P receptors. SCC-VII cells express the 46 kDa cation-dependent M6P receptor (MPR46), but intracellular retention of procathepsins B, D, and L is not affected by ammonium chloride and chloroquine, agents known to perturb the M6P receptor system. Our results indicate that failure to express the 300 kDa M6P/IGF2R may enhance the metastatic capacity of tumor cells by inducing the secretion of procathepsins B, D, and L.

Renal cell carcinoma (RCC) is an angiogenic tumor resistant to standard cytotoxic chemotherapeutic agents. Although often responsive to immunomodulatory agents including interleukin 2 and IFN-alpha, the overall results in randomized Phase III studies are disappointing with only modest improvements in overall survival. This Phase II study evaluated the efficacy and tolerability of razoxane, an antiangiogenic topoisomerase II inhibitor, in 40 patients (32 men, 8 women; age: range, 31-76 years; median, 58 years) with inoperable RCC. Twenty patients received razoxane 125 mg p.o., twice a day for 5 days each week for 8 weeks (one cycle). This was repeated in patients with stable disease (StD), but was discontinued after <em>16</em> weeks if there was no evidence of an objective response. Because minimal toxicity was seen, subsequent patients (n = 20) were treated until progressive disease (PD) was documented. Of 38 evaluable patients, 11 (29%) had StD for a minimum of 4 months, and the remainder had PD. Median overall survival was 7.3 months. Duration of survival was significantly better in patients with StD compared with those with PD (P = 0.003). The effect of treatment on six potential surrogate serum/plasma (vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), urokinase plasminogen activator soluble receptor (uPAsr), E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand's <em>factor</em> (vWF) and two urinary (VEGF and bFGF) markers of angiogenesis was evaluated before and after 1 cycle of treatment. Pretreatment serum VEGF and E-selectin levels above the median value were associated with a poor prognosis. Serum VCAM-1 levels and urinary VEGF levels rose significantly after one cycle in patients with PD but not in those with StD. Serum VEGF, bFGF, VCAM-1 and vWF, plasma uPAsr and urinary bFGF levels were significantly higher in PD patients compared with StD patients before and/or after 1 cycle of treatment. In conclusion, razoxane is an antiangiogenic agent that has minimal toxicity and that requires further evaluation in combination with other active agents in the treatment of RCC. Surrogate serum and urinary markers of angiogenesis may have a role to play in predicting disease response and overall survival in RCC.

We previously reported that a <em>16</em>-kDa proteolytic fragment of IGF Binding Protein-3 (IGFBP-3), which is devoid of affinity for IGFs, inhibits the mitogenic effects of IGF-I on chick embryo <em>fibroblasts</em>. Here, we set out to determine if the fragment had biological effects on <em>fibroblasts</em> from mouse embryos homozygous for a targeted disruption of the Type 1 IGF receptor gene. In the cell clone used, bFGF (but not IGF, EGF or PDGF) was mitogenic in serum-free medium, increasing 14C-thymidine uptake by a <em>factor</em> of 10-15 within 24 hours and doubling cell proliferation. The <em>16</em>-kDa fragment, isolated by HPLC following limited proteolysis of recombinant human (rh) IGFBP-3 by plasmin, in both assays dose-dependently (20 to 100 ng/ml) inhibited (up to 100%) maximal stimulation induced by 25 ng/ml bFGF, whereas intact IGFBP-3 had virtually no effect. Similar results were obtained with control wild-type cells. In the latter, the mitogenic activity of 1% fetal calf serum (equal to that of 25 ng/ml bFGF) was inhibited by only 25-30% by 100 ng/ml <em>16</em>-kDa fragment or 200 ng/ml rhIGFBP-3. This agrees with an antagonistic action, affecting the mitogenic activity of serum that is attributable to IGFs. The <em>16</em>-kDa IGFBP-3 fragment therefore appears to be a potent inhibitor of mitogenic signals resulting from activation of both the type 1 IGF and FGF receptors.

The bryostatins, a group of macrocyclic lactones isolated on the basis of their antineoplastic activity, protein kinase C in vitro and block phorbol ester binding to this enzyme. In some cellular systems, bryostatins mimic phorbol ester action. In other systems, however, the bryostatins display only marginal agonistic action and, instead, inhibit phorbol ester-induced responses. At least in primary mouse epidermal cells, a transient duration of action of bryostatin 1 could rationalize these differences. To determine whether this model of transient activation could explain the dual actions of bryostatin 1 in other cell systems, we have examined the effects of bryostatin 1 on short-term responses in C3H 10T1/2 mouse <em>fibroblasts</em>. Even at very short exposures (30 min), bryostatin 1 blocked phorbol ester-induced arachidonic acid metabolite release and induced only minimal release when assayed alone. In contrast, epidermal <em>growth</em> <em>factor</em> binding was markedly and rapidly decreased in bryostatin 1-treated C3H 10T1/2 cells, and this decrease showed only limited reversal <em>16</em> h after initial exposure. Bryostatins 2, 3, 4, 10, and several of their derivatives caused variable arachidonic acid metabolite release (10 to 60% of phorbol ester control) and correspondingly variable inhibition of phorbol ester action. Our findings on arachidonic acid metabolite release argue against transient activation of the protein kinase C pathway as the sole explanation of bryostatin 1 action. They indicate, moreover, differences in the structure-activity relations of the bryostatins for the phorbol ester-mimetic and phorbol ester-inhibitory actions.

To investigate the relationship between placenta <em>growth</em> <em>factor</em> (PlGF) and brain tumor angiogenesis, we screened 36 primary and 3 metastatic brain tumors. We examined the expression of PlGF mRNA with respect to vasculature of various tumors which was determined by preoperative angiography. The expression of genes of the other angiogenic <em>factors</em>, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was also tested, and compared to that of PlGF. The primary tumors consisted of <em>16</em> meningiomas, 7 gliomas, 7 schwannomas, 4 pituitary adenomas, 1 germinoma, and 1 choriocarcinoma. Using a quantitative reverse transcription-polymerase chain reaction, the mRNA for PlGF149 and PlGF170 were detected in 25 out of 39 (64.1%) brain tumors. In primary brain tumors, PIGF mRNA was expressed in all the hypervascular tumors, but only in 5 of <em>16</em> hypovascular tumors (31.3%). None of the 3 metastatic hypervascular tumors expressed PlGF mRNA. The VEGF and bFGF mRNA expression was both detected in 87.2% of the tumors examined. We conducted hypoxic experiments with cultured U-251MG human glioma cells to determine the mechanism of PIGF gene regulation. As the atmospheric oxygen concentration was decreased, the PIGF mRNA level in the U-251MG cells was markedly increased. These results suggest that PIGF may contribute to the pathogenesis of brain tumor angiogenesis.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a 38 Kda cysteine-rich, heparin-binding peptide that has been implicated in several normal and abnormal physiological processes. CTGF has been shown to be induced by transforming <em>growth</em> <em>factor</em>-beta. Previous studies in our pig model of skin wound healing showed a coordinate expression of transforming <em>growth</em> <em>factor</em>-beta and CTGF during the healing process. To better understand the function of CTGF during wound healing, normal porcine <em>fibroblasts</em> were isolated from skin samples from SPF Yorkshire pigs. At fourth passage the cells were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum and at 80% confluence the medium was replaced with supplemented serum-free medium. After a further 24 hours, cells were treated with 0, 10, 25, 50, 100, and 500 ng/ml of 38 Kda or <em>16</em>-20 Kda (C-terminal truncated form) recombinant expressed human CTGF for 24 hours or treated with 100 ng/ml for 0, 12, 24, and 48 hours. Subsequently, CTGF effects on cell DNA synthesis and mRNA levels for a subset of relevant molecules were assessed. The results showed that in cells treated with 38 Kda rhCTGF, mRNA levels for types I and III collagen, fibromodulin, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were significantly up-regulated, but mRNA levels for HSP47, decorin, biglycan, and versican were not significantly altered. mRNA levels for CTGF were also significantly increased, indicating autoregulation of expression. However, mRNA levels for transforming <em>growth</em> <em>factor</em>-beta, inteleukins 1 and 6, tumor necrosis <em>factor</em>-alpha, and nerve <em>growth</em> <em>factor</em> did not change. Interestingly, mRNA levels for the tissue inhibitors of metalloproteinase-1, -2, -3 and -4 were observed to significantly increase, but in contrast, mRNA levels for matrix metalloproteinases-1, -2, -9 were not significantly altered by exposure of the cells to the 38 Kda form of CTGF. In addition, DNA synthesis was augmented in the presence of 38 Kda rhCTGF. However, the truncated <em>16</em>-20 Kda form of rhCTGF appeared to have none of these effects on porcine <em>fibroblasts</em>. These results indicate that in order to induce changes in porcine <em>fibroblasts</em> a molecule with an intact C-terminal domain is required, and that CTGF regulates porcine <em>fibroblast</em> extracellular matrix molecule, <em>growth</em> <em>factor</em>, and proteinase inhibitor gene expression without apparently affecting matrix metalloproteinase mRNA levels. These findings suggest that CTGF contributes to the anabolic environment during skin wound healing via selective modulation of <em>fibroblast</em> proliferation and changes to gene expression.

Impaired vascularity and nerve degeneration are the most important pathophysiological abnormalities of diabetic polyneuropathy (DPN). Therefore, regeneration of both the vascular and nervous systems is required for the treatment of DPN. The neural crest (NC) is a transient embryonic structure in vertebrates that differentiates into a vast range of cells, including peripheral neurons, Schwann cells, and vascular smooth muscle cells. In this study, we investigated the ability of transplantation of NC-like (NCL) cells derived from aged mouse induced pluripotent stem (iPS) cells in the treatment of DPN. iPS cells were induced to differentiate into neural cells by stromal cell-derived inducing activity (SDIA) and subsequently supplemented with bone morphogenetic protein 4 to promote differentiation of NC lineage. After the induction, p75 neurotrophin receptor-positive NCL cells were purified using magnetic-activated cell sorting. Sorted NCL cells differentiated to peripheral neurons, glial cells, and smooth muscle cells by additional SDIA. NCL cells were transplanted into hind limb skeletal muscles of <em>16</em>-week streptozotocin-diabetic mice. Nerve conduction velocity, current perception threshold, intraepidermal nerve fiber density, sensitivity to thermal stimuli, sciatic nerve blood flow, plantar skin blood flow, and capillary number-to-muscle fiber ratio were evaluated. Four weeks after transplantation, the engrafted cells produced <em>growth</em> <em>factors</em>: nerve <em>growth</em> <em>factor</em>, neurotrophin 3, vascular endothelial <em>growth</em> <em>factor</em>, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. It was also confirmed that some engrafted cells differentiated into vascular smooth muscle cells or Schwann cell-like cells at each intrinsic site. The transplantation improved the impaired nerve and vascular functions. These results suggest that transplantation of NCL cells derived from iPS cells could have therapeutic effects on DPN through paracrine actions of <em>growth</em> <em>factors</em> and differentiation into Schwann cell-like cells and vascular smooth muscle cells.

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 <em>16</em> and 17 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>.