Mesenchymal stem cells (MSCs) favor cancer growth by facilitating immunosuppression status in tumor microenvironment. However, the function and mechanism of MSCs in initiating and developing prostate cancer remains to be fully understood. In this study, we first found that MSCs promoted prostate cancer (PCa) tumor growth in vivo and cell proliferation in vitro by using PCs cell strain RM-1. Both exogenous and endogenous MSCs could be recruited into the tumor microenvironment by using bone-marrow transplantation model. We further demonstrated that PDGF-BB produced by RM-1 cell promoted MSCs proliferation in vivo and in vitro, which was abrogated by Si-RNA specific to PDGF-BB. And inflammatory cytokines, such as interferon gamma, tumor necrosis factor alpha, and anti-inflammatory cytokine transformation growth factor alpha, further increased the ability of RM-1 to produce PDGF-BB. Overall, PCa cells produced PDGF-BB favors the proliferation of MSCs, which may elicit immunosuppressive function and enable PCa cells to escape from the immunity surveillance in tumor inflammatory microenvironment.

Low density lipoprotein receptor-related protein 1 (LRP1) is indispensable for embryonic development. Comparing different genetically engineered mouse models, we found that expression of Lrp1 is essential in the embryo proper. Loss of LRP1 leads to lethal vascular defects with lack of proper investment with mural cells of both large and small vessels. We further demonstrate that LRP1 modulates Gi-dependent sphingosine-1-phosphate (S1P) signaling and integrates S1P and PDGF-BB signaling pathways, which are both crucial for mural cell recruitment, via its intracellular domain. Loss of LRP1 leads to a lack of S1P-dependent inhibition of RAC1 and loss of constraint of PDGF-BB-induced cell migration. Our studies thus identify LRP1 as a novel player in angiogenesis and in the recruitment and maintenance of mural cells. Moreover, they reveal an unexpected link between lipoprotein receptor and sphingolipid signaling that, in addition to angiogenesis during embryonic development, is of potential importance for other targets of these pathways, such as tumor angiogenesis and inflammatory processes.

Lung fibrosis is a major medical problem for the aging population worldwide. Fibroblast migration plays an important role in fibrosis. Focal Adhesion Kinase (FAK) senses the extracellular stimuli and initiates signaling cascades that promote cell migration. This study first examined the dose and time responses of FAK activation in human lung fibroblasts treated with platelet derived growth factor BB (PDGF-BB). The data indicate that FAK is directly recruited by integrin β1 and the subsequent FAK activation is required for fibroblast migration on fibronectin. In addition, the study has identified that α5β1 and α4β1 are the major integrins for FAK-mediated fibroblast migration on fibronect. In contrast, integrins αvβ3, αvβ6, and αvβ8 play a minor but distinct role in fibroblast migration on fibronectin. FAK inhibitor significantly reduces PDGF-BB stimulated fibroblast migration. Importantly, FAK inhibitor protects bleomycin-induced lung fibrosis in mice. FAK inhibitor blocks FAK activation and significantly reduces signaling cascade of fibroblast migration in bleomycin-challenged mice. Furthermore, FAK inhibitor decreases lung fibrotic score, collagen accumulation, fibronectin production, and myofibroblast differentiation in in bleomycin-challenged mice. These data demonstrate that FAK mediates fibroblast migration mainly via integrin β1. Furthermore, the findings suggest that targeting FAK signaling is an effective therapeutic strategy against fibrosis.

Specific, high-affinity binding of FGF2 was evaluated in cultured skeletal muscle satellite cells from young (3- to 4-week-old) and adult (9- to 12-month-old) rats prior to the first division in culture. Specific binding of FGF2 was detected on satellite cells from young rats at 18 h postplating, the earliest time examined, but specific binding was not detectable until 42 h on satellite cells from old rats. This correlates well with the delayed entry into the cell cycle exhibited by adult satellite cells and with the ability of satellite cells from rats of these ages to proliferate in response to FGF2. Patterns of tyrosine phosphorylation in whole cell extracts, following stimulation by FGF2, indicated specific FGF2 phosphorylation of proteins of 150/145, 90, 42, and 35 kDa in cells from both age groups. Several growth factors were evaluated for their ability to stimulate early entry of adult satellite cells into the cell cycle, and none of the following growth factors were able to activate proliferation of these cells: FGF2, IGF-1, IGF-2, PDGF-BB, TGF-beta 1, or TGF-beta 2. In addition, specific binding of FGF2 to 48-h cultures of adult satellite cells was not stimulated by FGF2, IGF-1, IGF-2, PDGF-BB, or TGF-beta 2, and specific binding was significantly decreased (P < 0.05) by FGF2 and TGF-beta 2. Specific binding was significantly lower in cells treated with PDGF-BB than in cells treated with either form of IGF but was greater than in cells treated with FGF2 or TGF-beta 2. The results of these experiments suggest that expression of functional FGF receptors on the surface of satellite cells may represent an important step in the activation of quiescent satellite cells.

Vascular smooth muscle cells (VSMCs) play pivotal roles in the development of vascular diseases. While microRNAs are important in vascular pathologies, a few is known about their functional roles in VSMC phenotypes. We profiled microRNA expression in PDGF-BB treated VSMCs and found microRNA-146b-5p (miR-146b-5p) was upregulated. Inhibition of miR-146b-5p blocked in response to PDGF while reducing VSMC proliferation and migration. These studies implicate miR-146b-5p as necessary for PDGF-induced VSMC phenotype transition. Downstream miR-146b-5p targets modulating VSMC phenotypes will be further identified. Our study will help to understand the role of VSMCs in the pathology of vascular diseases.

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet-derived growth factor-BB (PDGF-BB) through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF-BB stimulated membrane ruffling, migration, and proliferation. PDGF-BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF-BB-stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF-BB-induced proliferation. Selective antagonism of ERK kinase inhibited PDGF-BB-induced ERK phosphorylation and proliferation, but did not affect PDGF-BB-stimulated migration. Concentrations of PDGF-BB that stimulated migration and proliferation did not influence myosin-dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF-BB-induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF-BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF-BB-stimulated migration, but not proliferation, ERKs mediate PDGF-induced proliferation, but not migration, and (3) increases in myosin-dependent contractility are not required for PDGF-BB-stimulated migration.

BACKGROUND

To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).

METHODS

We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.

RESULTS

ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.

CONCLUSIONS

We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.

Cell migration and matrix remodeling are key events in tissue repair and restructuring. Osteoblasts are responsible for the production of new bone matrix during bone remodeling. The activity of these cells can be modulated by a number of factors. The current study evaluated the hypothesis that cigarette smoke extract can alter repair and remodeling responses of human osteoprogenitor cells and osteoblast-like cells and, therefore, could explain one mechanism by which cigarette smoking leads to osteoporosis. Human osteoprogenitor cells were isolated from normal human bone marrow and maintained in culture under either control conditions or conditions that induced differentiation into osteoblast-like cells. Both cell types migrated toward fibronectin and PDGF-BB as chemoattractants. Neither responded to TGF-beta1. The osteoprogenitor cells were more active in their chemotactic response. The chemotactic response of both cell types was inhibited by cigarette smoke extract in a concentration-dependent manner. Both cell types, when cultured in three-dimensional native collagen gels maintained in floating culture, induced contraction of their surrounding matrices. Contraction was augmented by serum, PDGF-BB, and TGF-beta1. Osteoprogenitor cells were less active in inducing contraction than were osteoblast-like cells. Contraction of both cell types was inhibited by cigarette smoke extract. Cigarette smoke extract also inhibited the production of fibronectin by both cell types maintained in three-dimensional culture. Addition of exogenous fibronectin partially restored the ability of the cells to contract three-dimensional collagen gels. The current study demonstrates that cigarette smoke can interfere with the ability of bone cells to participate in repair and remodeling events. Such an effect may be one mechanism leading to the development of osteoporosis.

Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10-50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10-30 microM) MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migration in vitro and suggest the potential of targeting MAPK to inhibit RPE migration in vivo.

We test the hypothesis that the fibrinogen-thrombin formulation of fibrin sealant combined with fibroblasts and PDGF-BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full-thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 x 10(5) cells/wound) embedded in the fibrinogen component. At post-wounding day 7, there was no difference in the diluted vs. non-diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF-BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two-fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant.

Growth factors, transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), are critical components of the cutaneous wound healing process. Little is known, however, about the expression of these growth factors in normal flexor tendon healing. In this study, we wished to examine which of these growth factors are present at 10 days following tendon injury in a canine flexor tendon repair model. Using immunohistochemical analysis, we found positive staining for all growth factors in both timing groups. TGF-beta was detected around the repair site and proximal to it. PDGF-AA, PDGF-BB and VEGF appeared in the whole tendon section following repair. EGF, IGF and bFGF were not seen in tenocytes but were present in inflammatory cells surrounding the repair site. These findings provide evidence that TGF-beta, EGF, PDGF-AA, PDGF-BB, IGF, bFGF and VEGF are all expressed at 10 days after tendon injury but by different cell types and in different locations. The time course of growth factor expression is an important element in wound healing, and a better understanding of where and when such factors are expressed may help in the development of methods to manipulate this expression, accelerate healing, and reduce adhesions.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Corneal epithelial stem cells and transient amplifying cells are located in the limbal and corneal regions, respectively. In a serum-free medium with or without different cytokines, limbal fibroblasts consistently produced greater levels of keratinocyte growth factor (KGF) transcript and protein than corneal fibroblasts, whereas corneal fibroblasts produced greater levels of hepatocyte growth factor/ scatter factor (HGF/SF) transcript and protein than limbal fibroblasts. Expression of HGF/SF transcript and protein was up-regulated mildly by epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), or platelet-derived growth factor B (PDGF-BB) but markedly by interleukin-1 beta (IL-1 beta) and was more pronounced in limbal than in corneal fibroblasts. Expression of KGF transcript was down-regulated by EGF, TGF-alpha, and PDGF-BB, was markedly up-regulated by IL-1 beta, and was more pronounced in limbal than in corneal fibroblasts. Expression of KGF protein was up-regulated markedly by IL-1 beta and moderately by PDGF-BB, especially in limbal fibroblasts. TGF-beta 1 uniquely turned off transcript and protein expression of HGF/SF and KGF in corneal fibroblasts. Although its transcript levels were similarly down-regulated in limbal fibroblasts, KGF protein levels were paradoxically up-regulated by TGF-beta 1 when added alone or with TGF-alpha or IL-1 beta. These data indicate that KGF and HGF/SF, two fibroblast-derived epithelial mitogens, are expressed differentially by limbal and corneal fibroblasts and are modulated by cytokines activated during epithelial-mesenchymal interactions, suggesting that they may play a different role in modulating corneal epithelial stem cells and transient amplifying cells.

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.

Tissue regeneration into a three-dimensional scaffold requires the stimulation of blood vessel ingrowth. We have employed a freely interconnecting porous scaffold developed by us to determine the utility of a covalently bound heparin surface coating for the delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) in vivo. The heparin surface was shown to release VEGF far more rapidly than PDGF-BB in vitro (VEGF: 75 ng/h for 24 h; PDGF-BB: 86 pg/h for >7 days). In rat subcutaneous implants, at 10 days the heparin surface alone increased vessel ingrowth substantially (p<0.05 vs. unmodified scaffold), release of VEGF resulted in a further increase (p<0.05 vs. heparinized scaffold), whereas PDGF-BB had no additional effect. The increase induced by the combination of growth factors was similar to VEGF alone. After 2 months, PDGF-BB, but not VEGF delivery, resulted in a substantial increase in vascularization above that induced by heparin (p<0.05). At the longer time point the combination of growth factors was similar to PDGF-BB. However, only the combination of growth factors significantly elevated the number of ingrowing arterioles (p<0.05 vs. heparinized scaffold). Thus, the covalent modification of a porous scaffold with heparin allows for the differential release of VEGF and PDGF-BB that results in both a rapid and sustained increase in scaffold vascularization.

OBJECTIVE

CD248 (tumor endothelial marker 1/endosialin) is found on stromal cells and is highly expressed during malignancy and inflammation. Studies have shown a reduction in inflammatory arthritis in CD248-knockout (CD248(-/-) ) mice. The aim of the present study was to investigate the functional effect of genetic deletion of CD248 on bone mass.

METHODS

Western blotting, polymerase chain reaction, and immunofluorescence were used to investigate the expression of CD248 in humans and mice. Micro-computed tomography and the 3-point bending test were used to measure bone parameters and mechanical properties of the tibiae of 10-week-old wild-type (WT) or CD248(-/-) mice. Human and mouse primary osteoblasts were cultured in medium containing 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid to induce mineralization, and then treated with platelet-derived growth factor BB (PDGF-BB). The mineral apposition rate in vivo was calculated by identifying newly formed bone via calcein labeling.

RESULTS

Expression of CD248 was seen in human and mouse osteoblasts, but not osteoclasts. CD248(-/-) mouse tibiae had higher bone mass and superior mechanical properties (increased load required to cause fracture) compared to WT mice. Primary osteoblasts from CD248(-/-) mice induced increased mineralization in vitro and produced increased bone over 7 days in vivo. There was no decrease in bone mineralization and no increase in proliferation of osteoblasts in response to stimulation with PDGF-BB, which could be attributed to a defect in PDGF signal transduction in the CD248(-/-) mice.

CONCLUSIONS

There is an unmet clinical need to address rheumatoid arthritis-associated bone loss. Genetic deletion of CD248 in mice results in high bone mass due to increased osteoblast-mediated bone formation, suggesting that targeting CD248 in rheumatoid arthritis may have the effect of increasing bone mass in addition to the previously reported effect of reducing inflammation.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder marked by relentless fibrosis and damage of the lung architecture. A growing body of evidence now suggests that IPF progresses as a result of aberrant epithelial-fibroblast crosstalk. Injured epithelia are a major source of growth factors such as PDGF which guide resident fibroblasts to injury sites.

RESULTS

In this study, we utilized a novel co-culture system to investigate the effect of fibroblast phenotype on their response to epithelial injury. Fibroblasts from normal lungs (NHLF) responded to epithelial injury and populated the wound site forming a fibroblast plug/mechanical barrier which prevented epithelial wound closure. IPF fibroblasts were impaired in their response to epithelial injury. They also expressed reduced PDGFRα compared to NHLFs and were defective towards PDGF-AA mediated directional movement. Neutralization of PDGF-AA and pan-PDGF but not PDGF-BB reduced the injury response of NHLFs thereby preventing the formation of the mechanical barrier and promoting epithelial wound closure. Co-culture of epithelial cells with IPF fibroblasts led to marked increase in the levels of pro-fibrotic growth factors - bFGF and PDGF and significant depletion of anti-fibrotic HGF in the culture medium. Furthermore, IPF fibroblasts but not NHLFs induced a transient increase in mesenchymal marker expression in the wound lining epithelial cells. This was accompanied by increased migration and faster wound closure in co-cultures with IPF fibroblasts.

CONCLUSIONS

Our data demonstrate that the IPF fibroblasts have an aberrant repair response to epithelial injury.

BACKGROUND

The leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins constitute an integral membrane protein family that has three members: LRIG1, LRIG2, and LRIG3. LRIG1 negatively regulates growth factor signaling, but little is known regarding the functions of LRIG2 and LRIG3. In oligodendroglial brain tumors, high expression of LRIG2 correlates with poor patient survival. Lrig1 and Lrig3 knockout mice are viable, but there have been no reports on Lrig2-deficient mice to date.

RESULTS

Lrig2-deficient mice were generated by the ablation of Lrig2 exon 12 (Lrig2E12). The Lrig2E12-/- mice showed a transiently reduced growth rate and an increased spontaneous mortality rate; 20-25% of these mice died before 130 days of age, with the majority of the deaths occurring before 50 days. Ntv-a transgenic mice with different Lrig2 genotypes were transduced by intracranial injection with platelet-derived growth factor (PDGF) B-encoding replication-competent avian retrovirus (RCAS)-producing DF-1 cells. All injected Lrig2E12+/+ mice developed Lrig2 expressing oligodendroglial brain tumors of lower grade (82%) or glioblastoma-like tumors of higher grade (18%). Lrig2E12-/- mice, in contrast, only developed lower grade tumors (77%) or had no detectable tumors (23%). Lrig2E12-/- mouse embryonic fibroblasts (MEF) showed altered induction-kinetics of immediate-early genes Fos and Egr2 in response to PDGF-BB stimulation. However, Lrig2E12-/- MEFs showed no changes in Pdgfrα or Pdgfrβ levels or in levels of PDGF-BB-induced phosphorylation of Pdgfrα, Pdgfrβ, Akt, or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Overexpression of LRIG1, but not of LRIG2, downregulated PDGFRα levels in HEK-293T cells.

CONCLUSIONS

The phenotype of Lrig2E12-/- mice showed that Lrig2 was a promoter of PDGFB-induced glioma, and Lrig2 appeared to have important molecular and developmental functions that were distinct from those of Lrig1 and Lrig3.

BACKGROUND

Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model.

METHODS

Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry.

RESULTS

When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates.

CONCLUSIONS

The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.

Increased expression of PDGF-beta receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-beta receptor by cathepsin B, thus suggesting that the absence of PDGF-beta receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-beta receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).

Platelet-derived growth factor (PDGF-BB homodimer) and transforming growth factor-beta 1 (TGF-beta 1) are potent wound-healing hormones that accelerate incisional repair. To identify more precisely and quantitatively the stage(s) of wound healing influenced by growth-factor therapy, we investigated the three sequential tissue repair processes--inflammatory cell influx, intracellular procollagen type I (PC-I) synthesis, and collagen cross-linking--in recombinant growth factor--treated wounds. Using newly developed automated, quantitative image-analysis techniques, we observed that PDGF-BB markedly augmented the directed migration of macrophages into wounds during the first week after wounding and triggered and earlier and more sustained influx of PC-I--containing fibroblasts into the wound when compared with results in TGF-beta 1-treated or control wounds (p = 0.015 at day 2; p = 0.007 at day 21). In contrast, automated image analysis revealed TGF-beta 1-treated wound fibroblasts had a nearly twofold increase in intracellular levels of PC-I protein when compared with PDGF-BB-treated or control wound fibroblasts (p = 0.004 at day 4). However, the influence of TGF-beta 1 was transient, and the longer duration of PDGF-BB activity suggested a later influence, perhaps on the collagen remodeling phase, which is ultimately required for increased wound strength. To address this possibility, collagen cross-linking in growth factor-treated wounds was inhibited by beta-aminoproprionitrile (BAPN) treatment, and wound breaking strength was analyzed. Both PDGF-BB and TGF-beta 1 continued to enhance repair in BAPN-treated rats, indicating that they do not function primarily at the level of collagen cross-linking. Thus, PDGF-BB appears to enhance the inflammatory phase of wound healing to indirectly trigger PC-I synthesis, whereas TGF-beta 1 quantitatively enhances PC-I synthesis directly, accounting for their differing duration of activities within healing wounds.

Mechanisms underlying stimulation of platelet-derived growth factor (PDGF) beta-receptors expressed on connective tissue cells in human colorectal adenocarcinoma were investigated in this study. PDGF-AB/BB, but not PDGF receptors, was expressed by tumor cells in situ, as well as in tumor cell isolates of low passage from human colorectal adenocarcinoma. In an experimental co-culture system, conditioned medium from tumor cells only marginally activated PDGF beta-receptors expressed on fibroblasts. In contrast, co-culturing of the two cell types led to a marked PDGF beta-receptor activation. Functional PDGF-AB/BB was found to be associated with heparinase-I-sensitive components on the tumor cell surface. PDGF-AB/BB, isolated from heparinase-I-sensitive cell surface components, induced a marked activation of PDGF beta-receptors. Furthermore, co-culturing tumor cells together with fibroblasts led to a sustained activation of PDGF beta-receptors expressed on fibroblasts. Double immunofluorescence staining of tissue sections from human colorectal adenocarcinoma, combined with computer-aided image analysis, revealed that nonproliferating tumor cells were the predominant cellular source of PDGF-AB/BB in the tumor stroma. In addition, PDGF-AB/BB-expressing tumor cells were found juxtapositioned to microvascular cells expressing activated PDGF beta-receptors. Confocal microscopy revealed a cytoplasmic and cell-membrane-associated expression of PDGF-AB/BB in tumor cells situated in the stroma. In contrast, epithelial cells situated in normal or tumorous acinar structures revealed only a cell-membrane-associated PDGF-AB/BB expression. The is vitro and in situ results demonstrate that tumor cells not only facilitate but also have the ability to modulate connective tissue cell responsiveness to PDGF-AB/BB in a paracrine fashion, through direct cell-cell interactions in human colorectal adenocarcinoma.

OBJECTIVE

The severity of pulmonary dysfunction and subsequent development of chronic lung disease (CLD) in preterm neonates depends on several factors, among them oxygen administration. The aim of this report is to compare the effects of high-frequency, oscillatory ventilation (HFOV) versus synchronized, intermittent, mandatory ventilation (sIMV) on serum cytokine levels (IL-6, IL-8, IL-10, MCP-1, PDGF-BB, VEGF and TGF-beta1) and ventilator indices during the first week of life. Moreover, CLD development and several other outcomes were compared between the two groups.

METHODS

Randomized clinical trial.

METHODS

Third level NICU.

METHODS

40 preterm neonates with a gestational age between 24 and 29 weeks were randomly (20 per group) assigned to one of the two, above-mentioned ventilation strategies within 30 minutes of birth.

RESULTS

At 1, 3 and 5 days, neonates were monitored by means of ventilator indices and levels of seven pro-inflammatory or anti-inflammatory (pro-fibrotic) cytokines in serum. No clinical or biochemical differences were observed at baseline. The neonates assigned to HFOV benefited from early and sustained improvement in gas exchange, with earlier extubation and lower incidence of CLD, as compared to the neonates assigned to sIMV treatment, and showed a significant reduction of serum IL-6, IL-8 and IL-10 over time only when the HFOV treatment was administered. In addition, at days 3 and 5, the IL-6 levels were significantly lower in the HFOV group as compared to sIMV patients.

CONCLUSIONS

The results of this randomized clinical trial support the hypothesis that early use of HFOV, combined with an optimum volume strategy, has a beneficial effect, reducing serum levels of pro-inflammatory cytokines and consequently the acute phase leading to lung injury.

During acute lung injury, there is an outpouring of growth factors into the alveolar space that drive local repair and fibrosis. During the remodeling that follows the instillation of bleomycin via the trachea into the adult rat, at least two platelet-derived growth factor (PDGF)-like peptides are released sequentially into lung lining fluid. Groups of four to five animals were killed at 3, 6, 15, and 26 days after exposure to bleomycin and lungs lavaged with isotonic saline. PDGF-like peptides in epithelial lining fluid (ELF) were partially purified by cation exchange chromatography and concentrated. Isolated peptides were analyzed by immunoblotting to determine their molecular weight and immunologic identity. Western blots were probed with polyclonal antibodies to PDGF-BB and PDGF-AA. PDGF-like peptides of two distinct size classes (38-40 kD and 29 kD) were present in alveolar fluid from all rats with lung injury induced by bleomycin. No PDGF-like peptides were found in comparably prepared ELF from control animals. The 38-40 kD peptide was detected only with anti-PDGF-BB antibody; the 29 kD peptide was detected only with anti-PDGF-AA antibody. The presence of these two peptides varied independently with time after exposure to bleomycin. The 38-40 kD peptide was at peak levels at 3 to 6 days. In contrast, the 29 kD peptide was present at all times following injury but with far less variation over time. In parallel with these immunoassays for PDGF-like molecules, there was abundant growth-promoting activity for fibroblasts present in concentrated ELF during the course of injury.(ABSTRACT TRUNCATED AT 250 WORDS)