OBJECTIVE

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular growth and recruitment of perivascular mural cells. The purpose of the present study is to investigate the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB.

RESULTS

PDGF-BB increased vascular sprouting and branching of capillary-like structures in embryoid bodies as evaluated by computer-assisted analysis of CD31-positive cell structures. It also activated extracellular-regulated kinase 1,2 (ERK1,2) and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinase or PI 3-kinase. Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular Ca(2+) levels in differentiating ES cells expressing the PDGF receptor beta, an effect that was abolished in the presence of the intracellular Ca(2+) chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels in embryoid bodies as evaluated using the redox-sensitive dye H(2)DCF-DA. ROS generation was blunted in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin as well as in the presence of BAPTA, suggesting that ROS generation is regulated by intracellular Ca(2+) transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzenesulfonylfluoride and VAS2870, the free radical scavengers vitamin E, and N-(2-mercaptopropionyl)glycin as well as by BAPTA.

CONCLUSIONS

Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca(2+)-induced ROS generation, resulting in the activation of an ERK1,2-mediated signal transduction cascade.

The ability of dopamine D(4) and D(2) receptors to activate extracellular signal-regulated kinases (ERKs) 1 and 2 was compared using Chinese hamster ovary (CHO-K1) cells transfected with D(4.2), D(4.4), D(4.7), and D(2L) receptors. Dopamine stimulation of D(4) or D(2L) receptors produced a transient, dose-dependent increase in ERK1/2 activity. Receptor-specific activation of the ERK mitogen-activated protein kinase (MAPK) pathway was confirmed using the D(2)-like receptor-selective agonist quinpirole, whereas the specific antagonist haloperidol blocked activation. MAPK stimulation was dependent on a pertussis-toxin-sensitive G protein (G(i/o)). trans-Activation of the platelet-derived growth factor (PDGF) receptor was an essential step in D(4) and D(2L) receptor-induced MAPK activation. PDGF receptor-selective tyrosine kinase inhibitors tyrphostin A9 and AG1295 abolished or significantly inhibited ERK1/2 activation by D(4) and D(2L) receptors. Dopamine stimulation of the D(4) receptor also produced a rapid increase in tyrosine phosphorylation of the PDGF receptor-beta. The Src-family tyrosine kinase inhibitor PP2 blocked MAPK activation by dopamine; however, this drug was also found to inhibit PDGF-BB-stimulated ERK activity and autophosphorylation of the PDGF receptor-beta. Downstream signaling pathways support the involvement of a receptor tyrosine kinase. The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, protein kinase C inhibitors GF109203X and Calphostin C, dominant-negative RasN17, and the MEK inhibitor PD98059 significantly attenuated or abolished activation of MAPK by dopamine D(4) and D(2L) receptors. Our results indicate that D(4) and D(2L) receptors activate the ERK kinase cascade by first mobilizing signaling by the PDGF receptor, followed by the subsequent activation of ERK1/2 by pathways associated with this receptor tyrosine kinase.

The migration of arterial vascular smooth muscle cells (VSMC) is thought to play a central role in atherogenesis and restenosis. The migration of several other cell types, including monocytes, T-lymphocytes and endothelial cells is also involved in the development of the mature atherosclerotic lesion. Several defined growth factors, cytokines and extracellular matrix components which are released at the sites of lesions have been implicated in the regulation of migration of VSMC and other lesion-associated cells. Platelet-derived growth factor BB-homodimer of PDGF (PDGF-BB) is strongly implicated in neo-intima formation in vivo and is the most potent known chemoattractant for VSMC in vitro. Dynamic interactions between cell surface adhesive receptors (integrins) for ECM components, organisation of the actin cytoskeleton and the turnover of focal adhesions are all key processes in cell locomotion and migration. The signal transduction pathways which mediate the chemotactic effects of PDGF-BB and other migration factors on VSMC are unknown, but several classes of cellular components are implicated including components associated with focal adhesions, small GTP-binding proteins of the rho family, and certain substrates of the PDGF beta-receptor. Tyrosine phosphorylation of the novel focal adhesion-associated protein tyrosine kinase, p125 focal adhesion kinase (p125FAK), is regulated by integrins and by several factors which alter actin cytoskeletal organisation. Recent findings suggest that tyrosine phosphorylation of p125FAK and other focal adhesion-associated proteins may be implicated in the chemotactic response of VSMC to PDGF-BB. The migratory response to PDGF-BB may be dependent on both ligand isoform bio-availability and on receptor-isotype expression as well as on down-stream signalling events. Ultimately, cell migration in vivo will be determined by a complex array of diverse extracellular molecules organised in intercellular paracrine/autocrine networks as well as multiple interacting intracellular signal transduction pathways.

Tissue engineering of small-diameter blood vessels is still challenging because of restenosis and burst. To prevent thrombosis, rapid endothelialization along the lumen of grafts is intended, followed by proliferation of vascular smooth muscle cells (VSMCs) around the exterior for compliance. To this goal, two modified coaxial electrospinning techniques were developed to encapsulate vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGF), respectively, to regulate proliferation of vascular endothelial cells (VECs) and VSMCs. Release profiles, in vitro cell proliferation and in vivo implantation of double-layered electrospun membranes were investigated, and what made it special was the electrospun membranes were composed of chitosan hydrogel/poly(ethylene glycol)-b-poly(L-lactide-co-caprolactone) (PELCL) electrospun membrane loaded with VEGF as the inner layer and emulsion/PELCL electrospun membrane-loaded PDGF as the outer. It was found that dual-release of VEGF and PDGF could accelerate VEC proliferation in the first 6 days, and modulate slow VSMC proliferation in the initial 3 days whereas generate rapid proliferation after day 6, which is of great benefit to blood vessel regeneration. Four weeks of in vivo replacement of rabbit carotid artery demonstrated that VECs and VSMCs developed on the lumen and exterior of vascular grafts, respectively, and no thrombus or burst appeared. It was concluded that dual-delivery of VEGF and PDGF by the modified electrospun membranes could facilitate revascularization.

Through immunohistochemical studies we have identified the cell-surface proteoglycan, NG2, on blood vessels throughout the rat embryo. The particular cell type expressing this chondroitin sulfate proteoglycan, however, is dependent upon tissue location. Microvessels within the rat CNS express NG2 on endothelial cells, while in blood vessels outside the CNS, NG2 is found on smooth muscle cells. To analyze what role NG2 might play in these blood vessels, an enzymatic dissociation protocol was used to establish primary cultures of vascular smooth muscle cells from Postnatal Day 3 rat aorta. In this study we demonstrate the involvement of NG2 in the mitogenic and chemoattractant responses of smooth muscle cells to PDGF. In assays measuring either DNA synthesis or cell migration, treatment of smooth muscle cells with anti-NG2 immunoglobulins decreased their responses to PDGF-AA but had no effect upon their ability to react to PDGF-BB. These results support a role for NG2 in potentiating signaling through the alpha PDGF receptor in vascular smooth muscle cells. The presence of the proteoglycan on a large subpopulation of these cells could provide an enhanced response to the growth factor in times of active normal growth or in pathological conditions, such as arterial injury or atherosclerosis.

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.

Impaired wound healing results in significant morbidity for the surgical patient. The genetically diabetic (C57BL/KsJ-db/db) mouse is obese, hyperglycemic, insulin-resistant, and exhibits markedly impaired wound healing. Previous studies have demonstrated that the fibroblast mitogens, BB homodimer of platelet-derived growth factor (PDGF-BB) or basic fibroblast growth factor, plus insulin-like growth factor, act synergistically to enhance wound closure in the genetically diabetic mouse. The purpose of this study was to determine whether the keratinocyte mitogens, epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha), in combination with the fibroblast mitogen, PDGF-BB, would produce a similar synergistic enhancement in tissue repair. Full-thickness skin wounds created on the backs of diabetic mice received topical applications of vehicle (5% polyethylene glycol), PDGF-BB (10 micrograms), EGF (1 microgram), TGF-alpha (1 microgram), or the combination of PDGF (10 micrograms) and EGF (1 microgram) or TGF-alpha (1 microgram) for 5 consecutive days starting at wounding. Application of PDGF-BB or TGF-alpha alone to wounds in diabetic animals improved wound closure when compared to vehicle treatment. EGF did not affect healing and did not have any additive effects when combined with PDGF-BB. Significant improvements in wound closure were observed with the combination of PDGF-BB and TGF-alpha when compared to treatment with the individual growth factors. The PDGF-BB/TGF-alpha combination accelerated healing in the diabetic animals to a rate that was closer to that seen in nondiabetic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidic and basic fibroblast growth factors (aFGF, bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor type beta (TGF-beta) are well-characterized growth regulators. Though there are reports of the effects of some of these factors on vascular cells, variability in the purity of growth factor preparations and differing cell culture conditions make comparison among the results difficult. Thus, a study employing homogeneous preparations of recombinant growth factors on well-characterized cell populations was conducted. Each of the factors was tested for its effect on the proliferation of cells derived from large and small blood vessels: aortic and capillary endothelial cells (EC), smooth muscle cells (SMC) and pericytes. Of the five growth factors only bFGF stimulated the proliferation of both large vessel and microvessel EC; aFGF was mitogenic for microvessel but not large vessel EC. Neither PDGF (AA, BB or AB) nor EGF had any effect on EC growth. TGF-beta was a potent inhibitor of both aortic and capillary EC proliferation with half maximal inhibition at concentrations as low as 0.05 and 0.25 ng/ml for microvessel and aortic EC, respectively. The FGFs were potent mitogens for the mural cells, SMC and pericytes. Likewise, PDGF was stimulatory for SMC and pericytes. The BB homodimer yielded the greatest degree of growth stimulation for both pericytes and SMC. In the case of SMC the AA homodimer stimulated small but significant growth and the effect of AB was intermediate to that of the homodimeric forms. On the other hand, neither the AA homodimer nor the heterodimer stimulated any significant increase in pericyte number. EGF was moderately mitogenic for both types of mural cells, reaching maximum stimulation at 100 pg/ml of EGF. TGF-beta was inhibitory for SMC but not for pericyte proliferation. These data indicate both quantitative and qualitative differences between the responses of large and small vessel EC to the various growth factors and distinct differences in the responsiveness of SMC and pericytes to PDGF and TGF-beta.

BACKGROUND

Connective tissue growth factor (CTGF) is a profibrotic growth factor, which is upregulated in wound healing and renal fibrosis, including anti-Thy-1.1 nephritis. The kinetics of CTGF mRNA expression in anti-Thy-1.1 nephritis suggested that CTGF regulation might contribute to glomerular response to injury downstream of transforming growth factor-beta (TGFbeta). In anti-Thy-1.1 nephritis the initial damage is followed by mesangial repair and limited sclerosis, which involves mesangial cell (MC) activation (alpha-smooth-muscle actin (alphaSMA) expression), proliferation, migration, and extracellular matrix production. The present in vitro study addresses the possible role of CTGF in these different aspects of mesangial response to injury, and how CTGF activity might relate to effects of TGFbeta and platelet-derived growth factor-BB (PDGF-BB).

RESULTS

Immunostaining and ELISA showed that alphaSMA expression and transformation of MC into myofibroblast-like cells was induced by TGFbeta, but not affected by PDGF-BB, CTGF, or neutralizing anti-CTGF antibodies. [(3)H]thymidine incorporation and Ki67 staining demonstrated that, unlike PDGF-BB, neither CTGF nor TGFbeta induced the proliferation of MC. In contrast, both CTGF and TGFbeta induced MC migration, as evidenced by approximation of wound edges in scrape-wounded, non-proliferating rat MC monolayers. In addition, fibronectin expression was upregulated by both CTGF and TGFbeta, as measured by dot-blot analysis. Anti-CTGF completely blocked the effect of added CTGF. Moreover, anti-CTGF significantly reduced TGFbeta-induced increase in fibronectin.

CONCLUSIONS

It thus appears that CTGF is specifically involved in a subset of the adaptive changes of MC involved in mesangial repair and sclerosis, which makes it an interesting candidate target for future intervention strategies.

Migration of vascular smooth muscle cells (VSMCs) is a crucial response to vascular injury resulting in neointima formation and atherosclerosis. Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies in the vasculature. However, little is known about the intracellular pathways that mediate VSMC migration. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) activation in this function, since PDGF-BB as well as other growth factors activate this pathway. Using an in-gel kinase assay, we observed that PD 98059 an inhibitor of MEK that activates MAP kinase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 in cultured rat aortic smooth muscle cells in a concentration-dependent manner. In contrast, PDGF-mediated activation of intracellular calcium release was not affected by PD 98059. The chemotactic response of both rat aortic smooth muscle cells (RASMCs) and human umbilical vein smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was significantly reduced by PD 98059 (10 mumol/L) to 41.7 +/- 7.1% in RASMCs (P < .01) and to 47.2 +/- 5.3% in HUSMCs (P < .01). Similar inhibition was seen at 30 mumol/L, less at 1 mumol/L. To further confirm the specificity of these results implicating the MAPK pathway, an antisense oligodeoxynucleotide (ODN) directed against the initiation translation site of rat ERK-1 and ERK-2 mRNA was used to suppress MAP kinase synthesis and function in rat VSMCs. Liposomal transfection with 0.4 mumol/L antisense ODN reduced ERK-1 and ERK-2 protein by 65% (P < .01) after 48 hours. The chemotactic response to PDGF-BB (10 ng/mL) was reduced by 75% (P < .01) in rat VSMCs transfected with the same antisense ODN concentration. Sense and scrambled control ODNs (0.4 mumol/L) did not affect ERK-1 and ERK-2 protein concentrations or chemotaxis of VSMCs induced by PDGF-BB. These experiments provide the first evidence that activation of MAPK is a critical event in PDGF-mediated signal transduction regulating VSMC migration.

OBJECTIVE

Adhesion and migration of retinal pigment epithelial (RPE) cells to provisional extracellular matrices (ECM) is important in the development of epiretinal membranes found in proliferative vitreoretinopathy (PVR). Tumor necrosis factor alpha (TNF-alpha) is found in PVR membranes and regulates many functions of RPE cells. In this study, the effects of TNF-alpha on adhesion and migration of RPE cells to various components of ECM were examined and elucidation of the mechanism of the response was attempted.

METHODS

Mitogen activated protein kinase (ERK1/2; MAPK) activation was measured by immunoblot. RPE cells pretreated with TNF-alpha (10 ng/ml) or TNF-alpha + PD98059 (a specific inhibitor of MAPK, 30 microM) for 24 hours were compared with control RPE. Attachment was measured by modified MTT assay on fibronectin and collagen types I and IV. Spreading was measured by staining with fluo3-AM and confocal laser scanning microscopy. Migration of RPE cells on substrates was determined by Boyden chamber assay using PDGF-BB (20 ng/ml) as a chemotactic factor. Integrin expression was determined by flow cytometry and RT-PCR.

RESULTS

TNF-alpha rapidly activated MAPK and increased the extent of attachment, spreading and migration on fibronectin and collagen type I (P < 0.01) but not on collagen type IV. TNF-stimulated RPE cells showed increased mRNA and surface protein expression for alpha1 and alpha5 integrin (P < 0.01) but not alpha3 integrin subunit. Neutralizing the anti-alpha1 antibody inhibited migration on collagen type I, whereas alpha5 antibody inhibited fibronectin-induced migration. Treatment with both TNF and PD98095 reduced attachment and migration on provisional ECM and reduced the upregulated integrin expression to control levels.

CONCLUSIONS

After treatment with TNF-alpha, there is increased expression of specific integrins associated with increased adhesion and migration on provisional ECM (fibronectin and collagen type I). This effect is mediated, at least in part, by activation of MAPK signaling pathway.

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 x 10(8) or 5.5 x 10(9) pfu ml(-1)), Ad encoding luciferase (Ad-Luc; 5.5 x 10(9) pfu ml(-1); control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml(-1)). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, micro-computed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo.

Recent reports have described several cellular phenotypes that appear to be mediated by Endosialin/TEM-1/CD248 (TEM-1), including tubule formation on matrigel, migration and proliferation. It has been shown that siRNA knock-down of TEM-1 in primary human fibroblasts resulted in reduced proliferation. However, the downstream signaling events that mediate TEM-1 function(s) currently remain unknown. In this study, we demonstrate that TEM-1 mediates proliferation of primary human pericytes through a PDGF receptor signaling pathway. Normal pericytes expressing high levels of TEM-1 were able to proliferate, respond to PDGF-BB stimulation by phosphorylating both the PDGF receptor and the MAP kinase ERK-1/2, and induce the expression of the immediate early transcription factor c-Fos. However, when TEM-1 expression was knocked-down, PDGF-BB-induced proliferation, ERK-1/2 phosphorylation, and c-Fos expression were significantly impaired. Thus, our results provide evidence for a TEM-1-dependent signal pathway that controls proliferation of human pericytes and suggest targeting this pathway for future strategies aimed at mitigating tumor angiogenesis.

A significant problem in flexor tendon repair is the lack of suitable graft material for reconstruction. The ex vivo production of flexor tendon graft constructs requires the expansion of primary cells. Growth factors, such as platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF), are known to promote tendon healing and tendon cell proliferation. The purpose of these experiments was to optimize tenocyte proliferation in 3 tendon cell populations using growth factor supplementation. Cells of the synovial sheath, epitenon, and endotenon were isolated from rabbit flexor digitorum profundus tendons and maintained in culture. Cell cultures were supplemented with IGF-1, PDGF-BB, and bFGF alone and in combination. The conditions used for individual growth factor supplementation were IGF-1 (10, 50, and 100 ng/mL), PDGF-BB (1, 10, and 50 ng/mL), and bFGF (0.5, 1, and 5 ng/mL). The conditions used for combinations of growth factors were IGF-1 + PDGF-BB (50 + 10 and 100 + 50 ng/mL, respectively) and IGF-1 + PDGF-BB+ bFGF (50 + 10 + 1; 50 + 10 + 5; 100 + 50 + 1; and 100 + 50 + 5 ng/mL, respectively). For all 3 tendon cell populations, proliferation at 72 h was greater in the presence of individual growth factors as compared to controls. With PDGF-BB (50 ng/mL) supplementation, mean absorbance values increased 97% (0.57 to 1.13) in S cells, 37% (0.51 to 0.70) in E cells, and 33% (0.33 to 0.44) in T cells ( p < 0.001). In addition, a synergistic effect was observed. The combination of growth factors resulted in greater proliferation as compared to maximal doses of individual growth factors. In cultures supplemented with IGF-1 (100 ng/mL) +PDGF-BB (50 ng/mL), mean absorbance increased 114% (0.57 to 1.22) in S cells, 63% (0.51 to 0.831) in E cells, and 47% (0.33 to 0.48) in T cells ( p < 0.001). IGF-1 (100 ng/mL) + PDGF-BB (50 ng/mL) + bFGF (5 ng/mL) resulted in the greatest amount of cell proliferation for all 3 tendon cell populations. The mean absorbances increased 251% in S cells, 98% in E cells, and 106% in T cells ( p < 0.001). In summary, IGF-1, PDGF-BB, and bFGF can be used in combination to maximize tenocyte proliferation. Synergism among growth factors may provide a means to facilitate tendon engineering.

Rapid regrowth or recurrent growth of occult cancer cells are often observed after esophagectomy or postoperative complications. In order to clarify the mechanism of such oncological circumstances, we focused on neutrophil elastase (NE), which degrades a broad spectrum of extracellular matrix and cell surface proteins. In the present study, we demonstrated that NE stimulated the growth of all of the five esophageal cell lines (TE-1, -7, -8, -12 and -13) by MTT assay and promoted cell invasion by cell migration assay. Pro-transforming growth factor-alpha (pro-TGF-alpha) from the cell membrane was released to the culture medium as a mature form after treatment with 5 microg/ml NE, and it reached the maximum level of 153% compared to the control values at 15 min of treatment in TE-13 cells. The phosphorylation of epidermal growth factor receptor (EGFR) rapidly occurs after treatment with NE and triggered the extracellular signal-regulated kinases 1 and 2 (ERK) signaling pathway. Moreover, NE induced release of platelet-derived growth factor-AA (PDGF-AA), PDGF-BB and vascular endothelial growth factor (VEGF) to 141.9, 227.7, and 171.6% of the control values, respectively. A specific NE inhibitor, sivelestat, significantly inhibited the NE-induced cell proliferation, cell invasion and subsequently inhibited the signal transduction pathway. Furthermore, sivelestat significantly inhibited NE-induced release of TGF-alpha, PDGF-AA, PDGF-BB and VEGF in the medium in TE-13 esophageal carcinoma cells. These results strongly indicate that NE released from activated neutrophils stimulates the growth and progression of esophageal cancer cells by releasing the growth factors on the cell surface and that sivelestat, a specific NE inhibitor, blocks these processes. Furthermore, we postulate that postoperative administration of sivelestat might be useful as a new molecular-targeting cancer therapy as well as for the treatment of postoperative respiratory complications.

Candida albicans, the major invasive fungal pathogen of humans, can cause both debilitating mucosal infections and fatal invasive infections. Understanding the complex nature of the host-pathogen interaction in each of these contexts is essential to developing desperately needed therapies to treat fungal infections. RNA-seq enables a systems-level understanding of infection by facilitating comprehensive analysis of transcriptomes from multiple species (e.g., host and pathogen) simultaneously. We used RNA-seq to characterize the transcriptomes of both C. albicans and human endothelial cells or oral epithelial cells during in vitro infection. Network analysis of the differentially expressed genes identified the activation of several signaling pathways that have not previously been associated with the host response to fungal pathogens. Using an siRNA knockdown approach, we demonstrate that two of these pathways-platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9)-govern the host-pathogen interaction by regulating the uptake of C. albicans by host cells. Using RNA-seq analysis of a mouse model of hematogenously disseminated candidiasis (HDC) and episodes of vulvovaginal candidiasis (VVC) in humans, we found evidence that many of the same signaling pathways are activated during mucosal (VVC) and/or disseminated (HDC) infections in vivo. Our analyses have uncovered several signaling pathways at the interface between C. albicans and host cells in various contexts of infection, and suggest that PDGF BB and NEDD9 play important roles in this interaction. In addition, these data provide a valuable community resource for better understanding host-fungal pathogen interactions.

A significant stumbling block in the creation of functional three-dimensional (3D) engineered tissues is the proper vascularization of the constructs. Furthermore, in the context of electrospinning, the development of 3D constructs using this technique has been hindered by the limited infiltration of cells into their structure. In an attempt to address these issues, a hybrid mesh of poly (ɛ-caprolactone)-collagen blend (PCL/Col) and hyaluronic acid (HA) hydrogel, Heprasil™ was created via a dual electrodeposition system. Simultaneous deposition of HA and PCL/Col allowed the dual loading and controlled release of two potent angiogenic growth factors VEGF(165) and PDGF-BB over a period of five weeks in vitro. Furthermore, this manner of loading sustained the bioactivity of the two growth factors. Utilizing an in-house developed 3D co-culture assay model of human umbilical vein endothelial cells and lung fibroblasts, the growth factor-loaded hybrid meshes was shown to not only support cellular attachment, but also their infiltration and the recapitulation of primitive capillary network in the scaffold's architecture. Thus, the creation of a PCL/Col-Heprasil hybrid scaffold is a step forward toward the attainment of a 3D bio-functionalized, vascularized tissue engineering construct.

In sub-Saharan Africa, approximately 30 million pregnant women are at risk of contracting malaria annually. Nearly 36% of healthy pregnant women receiving routine antenatal care tested positive for Plasmodium falciparum HRP-II antigen in Ghana. We tested the hypothesis that asymptomatic HRP II positive pregnant women expressed a unique Th1 and Th2 phenotype that differs from healthy controls. Plasma from healthy (n = 15) and asymptomatic (n = 25) pregnant women were evaluated for 27 biomarkers (IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL- 17, Eotaxin, bFGF-2, G-CSF, GM-CSF, IFN-gamma, IP-10, MCP-1, MIP-1alpha, MIP-1beta, PDGF-bb, RANTES, TNF, and VEGF) associated with Th1 and Th2 cytokine homeostasis. IL-10 and G-CSF levels were elevated in the asymptomatic group when compared with the healthy group (P = .031 and .041, resp.). The median ratios of IL-1beta:5, IL-1beta:10, IL-1beta:G-CSF, IL-1beta:Eotaxin, IL-12:G-CSF, IL-15:10, IL-17:G-CSF, IL-17:Eotaxin, TNF:IL-4, TNF:IL-5, and TNF:G-CSF were significantly different among the two groups. Thus, asymptomatic malaria carriage may be linked to circulating levels of IL-10 and G-CSF.

The glycosaminoglycan hyaluronan is important in many tissuerepair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-kappaB (nuclear factor kappaB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-beta1 (transforming growth factor-beta1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [3H]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.

Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg-Gly-Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10-100 nM. FN-null cells cultured on recombinant CCBD (FNIII(8-11)) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII(8-11) contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII(8-11) and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration.

Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-beta transcripts than purified normal CD8(+) T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers--including alpha-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain--to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

Anti-platelet-derived growth factor (PDGF) drugs are routinely used in front-line therapy for the treatment of various cancers, but the molecular mechanism underlying their dose-dependent impact on vascular remodelling remains poorly understood. Here we show that anti-PDGF drugs significantly inhibit tumour growth and metastasis in high PDGF-BB-producing tumours by preventing pericyte loss and vascular permeability, whereas they promote tumour cell dissemination and metastasis in PDGF-BB-low-producing or PDGF-BB-negative tumours by ablating pericytes from tumour vessels. We show that this opposing effect is due to PDGF-β signalling in pericytes. Persistent exposure of pericytes to PDGF-BB markedly downregulates PDGF-β and inactivation of the PDGF-β signalling decreases integrin α1β1 levels, which impairs pericyte adhesion to extracellular matrix components in blood vessels. Our data suggest that tumour PDGF-BB levels may serve as a biomarker for selection of tumour-bearing hosts for anti-PDGF therapy and unsupervised use of anti-PDGF drugs could potentially promote tumour invasion and metastasis.

Migration and proliferation of smooth muscle cells (SMCs) are key events during neointimal formation in pathological conditions of vessels. Tenascin-C (TNC) is upregulated in the developing neointima of lesions. We evaluated the effects of TNC on responses of SMCs against platelet-derived growth factor (PDGF) stimulation. TNC coated on substrate promoted PDGF-BB-induced proliferation and migration of rat SMC cell line A10 in BrdU incorporation and transwell assays, respectively. Immunoblotting showed that TNC substrate enhanced autophosphorylation of PDGFR-β after PDGF-BB stimulation. Integrin αvβ3 is known to be a receptor for TNC in SMCs. In immunofluorescence and immunoblot of integrin αv subunit, clustering of αv-positive focal adhesions and upregulated αv expression were observed in the cells on TNC substrate. Immunoprecipitation using anti-integrin αvβ3 antibody demonstrated that PDGFR-β and integrin αvβ3 were co-precipitated and that the relative amount of PDGFR-β after the stimulation was increased by TNC treatment. TNC also promoted phosphorylation of focal adhesion kinase (FAK) at tyrosine (Y) 397 and Y925. The phosphorylated FAK was localized at focal adhesions in immunofluorescence. Phosphorylated SRC at Y418 was also seen at focal adhesions. Immunoprecipitation with αv antibody showed increased SRC association with the integrin signaling complex in the cells on TNC after PDGF treatment. In the cells on TNC substrate, crosstalk signaling between integrin αvβ3 and PDGFR-β could be amplified by SRC and FAK recruited to focal adhesions, followed by enhanced proliferation and migration of A10 cells by PDGF-BB.