Although PDGF family members play a vital role in cell proliferation, motility and chemotaxis via activation of structurally similar alpha- and beta-receptors, little is known of their function in ovarian regulation and induction of tumorigenesis. Microarray analyses of ovaries from young follitropin receptor knockout (FORKO) mice that are prone to late ovarian tumors upon aging have revealed significant imbalances in PDGF ligands and receptors. We hypothesized that FSH/FSH-R signaling may exert effects partly by regulation of PDGF the family. To further understand their implications for ovarian tumorigenesis, we studied FORKO ovaries and hormonal regulation of the PDGF family members in normal mice, by using RT-PCR, Q-PCR, immunohistochemistry and western blotting. While PDGF-C and PDGFR-alpha increased, PDGFR-beta mRNA and protein decreased significantly in absence of FSH-R signaling. In the normal ovary, PDGFR-alpha was not affected by gonadotropin (eCG) stimulation but PDGF-C and PDGFR-beta decreased. Administration of estradiol decreased PDGF and their receptors. To further probe the differential regulation of PDGF family members by eCG and estradiol, we co-administered eCG with estrogen antagonist, ICI 182780. Increase in PDGFR-alpha in the absence of estradiol suggests direct effects of FSH signaling. During the estrous cycle in mice PDGF-C, PDGF-D and PDGFR-alpha mRNA levels were higher at the proestrous. By IHC, we report for the first time the localization of PDGF-C, PDGFR-alpha and PDGFR-beta protein in mouse ovarian compartments including the surface epithelium that is also altered in mutants. Immunostaining of PDGFRs increased as the follicle developed to preantral stage and declined thereafter. Thus, FSH modulates PDGF family members, partly via E2, suggesting that loss of FSH-R signaling causes an imbalance of PDGF family members predisposing the abnormal ovarian follicular environment for inducing tumorigenesis in aging FORKO mice.

The female reproductive system (FRS) has a great capacity for regeneration. The existence of somatic stem cells (SSC) that are likely to reside in distinct tissue compartments of the FRS is anticipated. Normal SSC are capable of regenerating themselves, produce a progeny of cells that differentiate and maintain tissue architecture and functional characteristics, and respond to homeostatic controls. Among those SSC of the FRS that have been identified are: a) undifferentiated cells capable of differentiating into thecal cells and synthesizing hormones upon transplantation, b) ovarian surface epithelium stem cells, mitotically responsive to ovulation, c) uterine endometrial and myometrial cells, as clonogenic epithelial and stromal cells, and d) epithelial and mesenchymal cells with self-renewal capacity and multipotential from cervical tissues. Importantly, these cells are believed to significantly contribute to the development of different pathologies and tumors of the FRS.It is now widely accepted that cancer stem cells (CSC) are at the origin of many tumors. They are capable of regenerating themselves, produce a progeny that will differentiate aberrantly and do not respond adequately to homeostatic controls. Several cell surface antigens such as CD44, CD117, CD133 and MYD88 have been used to isolate ovarian cancer stem cells. Clonogenic epithelial and stromal endometrial and myometrial cells have been found in normal and cancer tissues, as side population, label-retaining cells, and CD146/PDGF-R beta-positive cells with stem-like features. In summary, here we describe a number of studies supporting the existence of somatic stem cells in the normal tissues and cancer stem cells in tumors of the human female reproductive system.

The potential role of p38 mitogen-activated protein (MAP) kinase in platelet-derived growth factor receptor-alpha (PDGF-Ralpha) gene expression was investigated using cultured rat pulmonary myofibroblasts. p38 MAP kinase was constitutively expressed in myofibroblasts and activated by interleukin (IL)-1beta. A pyridinylimidazole compound, SB203580, completely inhibited the ability of p38 MAP kinase activity to phosphorylate PHAS-1 substrate. SB203580 inhibited IL-1beta-induced up-regulation of PDGF-Ralpha mRNA and protein in a concentration-dependent manner. Other kinase inhibitors, including the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PDPDGF-Ralpha. The IL-1beta-induced increase in the number of (125)I-PDGF-AA-binding sites at the cell surface was reduced >70% by pretreatment with SB203580. Accordingly, an enhancement of PDGF-AA-stimulated DNA synthesis following IL-1beta pretreatment was blocked >70% by SB203580. SB203580 did not affect IL-1beta-induced ERK activation, yet enhanced IL-1beta-induced JNK activation approximately 2-fold. Treatment of cells with SB203580 after inhibition of transcription by actinomycin D decreased the half-life of IL-1beta-induced PDGF-Ralpha mRNA from >4 to approximately 1.5 h. Moreover, pretreatment of cells with cycloheximide blocked induction of PDGF-Ralpha mRNA by IL-1beta, suggesting that de novo protein synthesis was required for PDGF-Ralpha mRNA stabilization. These data indicate that p38 MAP kinase regulates PDGF-Ralpha expression at the translational level by signaling the synthesis of an mRNA-stabilizing protein.

OBJECTIVE

The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the alpha and beta PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C.

RESULTS

PDGF C growth factor domain (GFD) was shown to preferentially bind and activate alpha PDGFR and activate beta PDGFR when it is co-expressed with alpha PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in alpha granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation.

CONCLUSIONS

Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.

Angiotensin II can cause hypertension through enhanced vasoconstriction of renal vasculature. One proposed mechanism for reduction of angiotensin II-induced hypertension is through inhibition of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade. MEK/ERK has been shown to phosphorylate the regulatory subunit of myosin light chain at identical positions as myosin light chain kinase. There are multiple mechanisms proposed regarding angiotensin II-mediated ERK activation. We hypothesized that renal microvascular smooth muscle cells (RmuVSMCs) signal through a unique pathway compared with thoracic aorta smooth muscle cells (TASMCs), which is involved in blood pressure regulation. Use of epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptor-specific inhibitors 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and 6,7-dimethoxy-3-phenylquinoxaline (AG1296), respectively, demonstrates that angiotensin II activates ERK in TASMCs, but not RmuVSMCs, through transactivation of EGF and PDGF receptors. In addition, inhibition of Src with its specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2) abolishes angiotensin II-, but not EGF-or PDGF-, mediated phosphorylation of ERK in RmuVSMCs, yet it has no effect in TASMCs. The physiological significance of transactivation was examined in vivo using anesthetized Wistar-Kyoto rats with 15 mg/kg 2'-amino-3'-methoxyflavone (PD98059), an MEK inhibitor, as well as 20 mg/kg AG1478 and 1.5 mg/kg AG1296 in an acute model of angiotensin II-mediated increase in blood pressure. None of the inhibitors had an effect on basal blood pressure, and only PD98059 reduced angiotensin II-mediated increase in blood pressure. Moreover, in RmuVSMCs, but not TASMCs, angiotensin II localizes phosphorylated ERK to actin filaments. In conclusion, angiotensin II signals through a unique mechanism in the renal vascular bed that may contribute to hypertension.

PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.

OBJECTIVE

To investigate the messenger RNA (mRNA) and protein expression of the recently discovered platelet-derived growth factor C (PDGF-C) and PDGF-D in the synovial membrane (SM) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to assess the localization and cellular source of these proteins in the SM and their functional influence on synovial fibroblasts.

METHODS

Expression of mRNA for PDGFs A, B, C, and D as well as for PDGF receptor (PDGFR) alpha and beta chains in RA and OA SM samples was assessed by real-time reverse transcription-polymerase chain reaction. Protein levels of PDGF-C and PDGF-D were quantified by immunoblotting. Regional and cellular localization of PDGF-C and PDGF-D in the SM was investigated by double-staining immunohistochemistry. In addition, the influence of PDGF-D on the proliferation of synovial fibroblasts and their matrix metalloproteinase (MMP-1) mRNA expression were determined.

RESULTS

The expression of mRNA for PDGFs A, B, and C and for PDGFR alpha and beta chains was comparable in RA and OA SM samples; in contrast, the expression of mRNA for PDGF-D was significantly higher in OA SM. PDGF-C protein was not differentially expressed in OA and RA. The expression of PDGF-D protein was significantly higher in RA SM (full-length and activated form). PDGF-C and PDGF-D were expressed throughout the SM (lining layer, diffuse infiltrates, and stroma) by both synovial fibroblasts and macrophages. In addition, PDGF-D increased the proliferation of synovial fibroblasts and the expression of mRNA for MMP-1.

CONCLUSIONS

PDGF-C and PDGF-D are expressed by synovial fibroblasts and macrophages in RA and OA SMs. The levels of PDGF-D protein were significantly higher in RA SM. In addition, PDGF-D stimulated synovial fibroblast proliferation and expression of MMP-1. These findings may have pathogenetic implications for cellular transformation and matrix remodeling in the RA SM.

Lymphangiogenesis (formation of new lymphatic vessels), unlike angiogenesis, has been a lesser-focused field in cancer biology, because of earlier controversy regarding whether lymphatic metastasis occurs via pre-existing or newly formed lymphatics. Recent evidence reveals that peri-tumoral or intra-tumoral lymphangiogenesis is a precursor for lymphatic metastasis in most carcinomas and melanomas. Two major lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, are produced by cancer cells or immune cells such as macrophages in the tumor-stroma to promote sprouting of lymphatics from lymphatic endothelial cells (LEC) or LEC precursors (LECP) by binding to their primary (high affinity) receptor VEGF-R3 or secondary receptors VEGF-R2, neuropilin (NRP)2 and α9/β1 integrin. Many other growth factors/receptors such as VEGF-A/VEGF-R2, fibroblast growth factor (FGF)2/FGF-R, platelet-derived growth factor (PDGF)/PDGF-R, hepatocyte growth factor (HGF)/C-Met, angiopoietins (Ang)1, 2/Tie2, and chemokines/ chemokine receptors (CCL21/CCR7, CCL12/CCR4) can also stimulate LEC sprouting directly or indirectly. This review deals with the roles of prostaglandins (PG), in particular PGE2, in cancer-associated lymphangiogenesis, with special emphasis on breast cancer. We show that cyclooxygenase (COX)-2 expression by breast cancer cells or tumor stroma leading to high PGE2 levels in the tumor milieu promotes lymphangiogenesis and lymphatic metastases, resulting from binding of PGE2 to PGE receptors (EP, in particular EP4) on multiple cell types: tumor cells, tumor-infiltrating immune cells, and LEC. EP4 activation on cancer cells and macrophages upregulated VEGF-C/D production to stimulate LEC sprouting. Furthermore, ligation of EP4 with PGE2 on cancer or host cells can initiate a new cascade of molecular events leading to cross-talk between cancer cells and LEC, facilitating lymphangiogenesis and lympho-vascular transport of cancer cells. We make a case for EP4 as a potential therapeutic target for breast cancer.

Cardiac repair and remodeling occur following myocardial infarction (MI). Our previous study demonstrated that platelet-derived growth factor (PDGF)-A/-D and PDGF receptors (PDGFR) are increased in the infarcted heart, with cells expressing PDGFR primarily endothelial and fibroblast-like cells. In the present study, we tested the hypothesis that PDGF contributes to cardiac angiogenesis and fibrogenesis post-MI. Rats with experimental MI were treated with either a PDGFR antagonist (Imatinib, 40 mg/kg/day) or vehicle by gavage, and sham-operated rats served as the controls. Cardiac fibrogenesis, angiogenesis, and ventricular function were detected at weeks 1 and 4 post-MI. We found that (1) transforming growth factor (TGF)-β1, tissue inhibitors of metalloproteinases (TIMP)-1/-2, and type I collagen mRNA were all significantly increased in the infarcted heart at week 1 post-MI, while PDGFR blockade significantly reduced these fibrogenic mediators in the noninfarcted myocardium as compared to controls; (2) fibrosis developed in both the infarcted and noninfarcted myocardium at week 4 with PDGFR blockade significantly suppressing collagen volume in the noninfarcted myocardium; (3) angiogenesis was activated in the infarcted myocardium, particularly at week 1, and was not altered by treatment with imatinib; and (4) ventricular dysfunction was evident in MI rats at week 4, and mildly improved with imatinib treatment. These observations indicated that PDGF can contribute to the development of cardiac interstitial fibrosis in the noninfarcted myocardium, but does not alter scar formation in the infarcted myocardium. Further, this study suggests the potential therapeutic effects of PDGFR blockade on interstitial fibrosis of the infarcted heart.

Screening of a compound library led to the identification of 2-amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor (PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replacement of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino group yielded 2a, which had greatly increased activity against all three TKs. In the present work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3-d]pyrimidine core provided several analogues that retained potency, including derivatives that were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which had IC50 values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively, was active in a variety of PDGF-dependent cellular assays and blocked the in vivo growth of three PDGF-dependent tumor lines.

Although the mechanisms of action by which aspirin, clopidogrel and dipyridamole inhibit platelets are well characterised, their effects on soluble modulators of thrombosis, inflammation, and endothelial function have yet to assessed systematically. In this investigation aspirin (A), clopidogrel (C), and dipyridamole (D) were administered singly and in combination (A, C, D, AC, AD, CD, ACD) in random order for 2 weeks (without washout) to 11 healthy subjects and 11 patients with previous ischaemic stroke. At the end of each treatment period plasma cyclic guanosine monophosphate (cGMP), monocyte chemoattractant pertide-1 (MCP-1), nitric oxide metabolites (NO(x)), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWf); and serum C-reactive protein (CRP) and platelet derived growth factor (PDGF); were measured blinded to treatment. Dipyridamole reduced plasma vWf levels (%) in both volunteers, -10.0 (4.95), and patients, -10.11 (4.34) (p < 0.05). Dipyridamole also lowered CRP (mg/l) in patients, -0.96 (0.47), but not volunteers. Clopidogrel reduced PAI-1 (ng/ml) in volunteers, -5.30 (2.20) (p < 0.05), and patients, -3.61 (2.75) (non-significant trend). Aspirin lowered PDGF (ng/ml) in volunteers, -3.46 (1.55), but not patients. Triple antiplatelet therapy was superior to dual and mono therapy in reducing vWf levels. In conclusion, antiplatelet agents have non-platelet-related effects on soluble modulators of thrombosis, inflammation, and endothelial function. In particular, dipyridamole reduces plasma vWf and clopidogrel lowers plasma PAI-1 levels. These effects may explain, in part, their roles in preventing atherothrombogenesis.

Signaling through platelet-derived growth factor receptor-α (PDGFRα) is required for alveolar septation and participates in alveolar regeneration after pneumonectomy. In both adipose tissue and skeletal muscle, bipotent pdgfrα-expressing progenitors expressing delta-like ligand-1 or sex-determining region Y box 9 (Sox9) may differentiate into either lipid storage cells or myofibroblasts. We analyzed markers of mesenchymal progenitors and differentiation in lung fibroblasts (LF) with different levels (absent, low, or high) of pdgfrα gene expression. A larger proportion of pdgfrα-expressing than nonexpressing LF contained Sox9. Neutral lipids, CD166, and Tcf21 were more abundant in LF with a lower compared with a higher level of pdgfrα gene expression. PDGF-A increased Sox9 in primary LF cultures, suggesting that active signaling through PDGFRα is required to maintain Sox9. As alveolar septation progresses from postnatal day (P) 8 to P12, fewer pdgfrα-expressing LF contain Sox9, whereas more of these LF contain myocardin-like transcription factor-A, showing that Sox9 diminishes as LF become myofibroblasts. At P8, neutral lipid droplets predominate in LF with the lower level of pdgfrα gene expression, whereas transgelin (tagln) was predominantly expressed in LF with higher pdgfrα gene expression. Targeted deletion of pdgfrα in LF, which expressed tagln, reduced Sox9 in α-actin (α-SMA, ACTA2)-containing LF, whereas it increased the abundance of cell surface delta-like protein-1 (as well as peroxisome proliferator-activated receptor-γ and tcf21 mRNA in LF, which also expressed stem cell antigen-1). Thus pdgfrα deletion differentially alters delta-like protein-1 and Sox9, suggesting that targeting different downstream pathways in PDGF-A-responsive LF could identify strategies that promote lung regeneration without initiating fibrosis.

Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.

The emphasis on periodontal regeneration has been shifted towards the harmonization of bioactive molecules and physiological phases during regeneration. This study investigated whether the combination and sequential-release of platelet-derived growth factor (PDGF, mitogen) and simvastatin (differentiation factor) facilitated periodontal regeneration. PDGF and simvastatin were encapsulated in double-walled poly-( d,l-lactide) and poly-(d,l-lactide-co-glycolide) (PDLLA-PLGA) microspheres using the co-axial electrohydrodynamic atomization technique. Critical-sized periodontal defects on rat maxillae were filled with microspheres encapsulating BSA-in-core-shell (BB), PDGF-in-shell (XP), simvastatin-in-core and BSA-in-shell (SB), simvastatin-in-core and PDGF-in-shell, or unfilled with microspheres (XX), and examined at 14 and 28 days post-operatively. The resultant microspheres were around 15 μm diameter with distinct core-shell structure, and the fast-release of PDGF followed by slow-release of simvastatin was noted in the SP group. The SP group demonstrated significantly greater bone volume fraction and decreased trabecular separation compared to the XX group at day 14, and milder inflammatory cells infiltration and elevated tartrate-resistant acid phosphatase level were noted at day 28. Fibers were also well-aligned and obliquely inserted onto the root surface similar to native periodontal ligament with signs of cementogenesis in the SP group. In conclusion, the combination and sequential-release of PDGF-simvastatin accelerates the regeneration of the periodontal apparatus.

Platelet-derived growth factor-D (PDGF-D) is a more recent recognized growth factor involved in the regulation of several cellular processes, including cell proliferation, transformation, invasion, and angiogenesis by binding to and activating its cognate receptor PDGFR-β. After bile duct ligation or in the carbon tetrachloride-induced hepatic fibrosis model, PDGF-D showed upregulation comparable to PDGF-B. Moreover, adenoviral PDGF-D gene transfer induced hepatic stellate cell proliferation and liver fibrosis. We here investigated the molecular mechanism of PDGF-D involvement in liver fibrogenesis. Therefore, the GRX mouse cell line was stimulated with PDGF-D and evaluated for fibrotic markers and PDGF-D signaling pathways in comparison to the other PDGF isoforms. We found that PDGF-D failed to enhance Col I and α-smooth muscle actin (α-SMA) production but has capacity to upregulate expression of the tissue inhibitor of metalloprotease 1 (TIMP-1) resulting in attenuation of MMP-2 and MMP-9 gelatinase activity as indicated by gelatinase zymography. This phenomenon was restored through application of a PDGF-D neutralizing antibody. Unexpectedly, PDGF-D incubation decreased both PDGFR-α and -β in mRNA and protein levels, and PDGF-D phosphorylated typrosines specific for PDGFR-α and -β. We conclude that PDGF-D intensifies fibrogenesis by interfering with the fibrolytic activity of the TIMP-1/MMP system and that PDGF-D signaling is mediated through both PDGF-α and -β receptors.

MSC (mesenchymal stromal cells) can differentiate into renal adult cells, and have anti-inflammatory and immune-modulating activity. In the present study, we investigated whether MSC have protective/reparative effects in anti-Thy1 disease, an Ab (antibody)-induced mesangiolysis resulting in mesangioproliferative nephritis. We studied five groups of rats: (i) rats injected with anti-Thy1.1 Ab on day 0 (group A); (ii) rats injected with anti-Thy1.1 Ab on day 0+MSC on day 3 (group B); (iii) rats injected with anti-Thy1.1 Ab on day 0+mesangial cells on day 3 (group C); (iv) rats injected with saline on day 0+MSC on day 3 (group D); and (v) rats injected with saline on day 0 (group E). Rats were killed on days 1, 3, 7 and 14. MSC prevented the increase in serum creatinine, proteinuria, glomerular monocyte influx and glomerular histopathological injury. Furthermore, MSC suppressed the release of IL-6 (interleukin-6) and TGF-β (transforming growth factor-β), modulated glomerular PDGF-β (platelet-derived growth factor-β), and reset the scatter factors and their receptors, potentiating HGF (hepatocyte growth factor)/Met and inactivating MSP (macrophage-stimulating protein)/Ron (receptor origin nantaise). Few MSC were found in the kidney. These results indicate that MSC improve anti-Thy 1 disease not by replacing injured cells, but by preventing cytokine-driven inflammation and modulating PDGF-β and the scatter factors, i.e. systems that regulate movement and proliferation of monocytes and mesangial cells.

Introduction: Investigational anti-VEGF treatments for neovascular age-related macular degeneration (nAMD) aim to improve visual outcomes and reduce treatment burden; these include long-acting agents, combination strategies, topical agents, sustained-release, and genetic therapies. Areas covered: The authors provide a comprehensive review of investigational therapies for nAMD, focusing on therapies currently in clinical trial. Expert opinion: Long-acting anti-VEGF agents have demonstrated promising results in phase 3 studies, and include Brolucizumab, a single-chain antibody fragment, and Abicipar, a designed ankyrin repeat protein (DARPin). Other unique anti-VEGF agents in current trials include Conbercept - a fusion protein of the VEGF receptor domains, KSI-301 - an anti-VEGF antibody biopolymer conjugate, and OPT-302 - an inhibitor of VEGF-C/D. Strategies to activate the Tie-2 receptor, some in combination with VEGF inhibition, are of interest, with recent trials of Faricimab, ARP-1536, and nesvacumab. Topical anti-VEGF ± anti-PDGF agents, such as pazopanib, squalamine lactate, regorafenib, and LHA510 have shown limited efficacy and/or have not been advanced, although PAN-90806 continues to advance with promising initial results. Sustained-release anti-VEGF treatments, to address treatment burden, include the ranibizumab Port Delivery System, GB-102, NT-503, hydrogel depot, Durasert, and ENV1305. Similarly, genetic therapies, including RGX-314 and ADVM-022, aim to provide sustained anti-VEGF expression from the retina.

BACKGROUND

Wound healing is a complex process involving interactions among a variety of different cell types. The normal wound repair process consists of three phases--inflammation, proliferation, and remodeling that occur in a predictable series of cellular and biochemical events. Wounds are classified according to various criteria: etiology, lasting, morphological characteristics, communications with solid or hollow organs, the degree of contamination. In the last few years many authors use the Color Code Concept, which classifies wounds as red, yellow and black wounds. This paper presents conventional methods of local wound treatment (mechanical cleansing, disinfection with antiseptic solutions, wound debridement--surgical, biological and autolytic; wound closure, topical antibiotic treatment, dressing), as well as general measures (sedation, antitetanous and antibiotic protection, preoperative evaluation and correction of malnutrition, vasoconstriction, hyperglycemia and steroid use, appropriate surgical technique, and postoperative prevention of vasoconstriction through pain relief, warming and adequate volume resuscitation).

UNASSIGNED

Growth factors play a role in cell division, migration, differentiation, protein expression, enzyme production and have a potential ability to heal wounds by stimulating angiogenesis and cellular proliferation, affecting the production and the degradation of the extracellular matrix, and by being chemotactic for inflammatory cells and fibroblasts. There are seven major families of growth factors: epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), interleukins (ILs), and colony-stimulating factor (CSF). Acute wounds contain many growth factors that play a crucial role in the initial phases of wound healing. The events of early wound healing reflect a finely balanced environment leading to uncomplicated and rapid wound healing. Chronic wounds, for many reasons, have lost this fine balance. Multiple studies have evaluated the effect that exogenously applied growth factors have on the healing of chronic wounds. In the study conducted by Knighton and colleagues, topical application of mixture of various growth factors (PDGF, TGF-beta, PDAF, PF4, PDEGF) demonstrated increased wound healing over controls. Brown and associates demonstrated a decrease in skin graft donor site healing time of 1 day using topically applied EGF. Herndon and ass. used systemic growth hormone in burned children and reduction in healing time made a significant clinical difference by allowing earlier wound coverage and decreasing the duration of hospitalization. The TGF family of growth factors is believed to be primarily responsible for excessive scar formation, especially the beta 1 and beta 2 isoforms. TGF-beta 3 isoform has recently been described and may have an inhibitory function on scar formation by being a natural antagonist to the TGF-beta 1 and TGF-beta 2 isoforms. Cytokines, especially interferon-alpha (INF-alpha), INF-alpha, and INF-alpha 2b, may also reduce scar formation. These cytokines decrease the proliferation rate of fibroblasts and reduce the rate of collagen and fibronectin synthesis by reducing the production of mRNA. Expression of nitric oxide synthase (NOS) and heat shock proteins (HSP) have an important role in wound healing, as well as trace elements (zinc, copper, manganese). Applications of some drugs (antioxidants--asiaticoside, vitamin E and ascorbic acid; calcium D-pantothenate, exogenous fibronectin; antileprosy drugs--oil of hydnocarpus; alcoholic extract of yeast) accelerate wound healing. Thymic peptide thymosin beta 4 (T beta 4R) topically applicated, increases collagen deposition and angiogenesis and stimulates keratinocyte migration. Thymosin alpha 1 (T alpha 1R), peptide isolated from the thymus, is a potent chemoattractant which accelerates angiogenesis and wound healing. On the contrary, steroid drugs, hemorrhage and denervation of wounds have negative effect on the healing process.

Platelet-derived growth factor (PDGF)-D is a PDGF receptor β (PDGFRβ)-specific ligand implicated in a number of pathological conditions, such as cardiovascular disease and cancer, but its biological function remains incompletely understood. In this study, we demonstrate that PDGF-D binds directly to neuropilin 1 (NRP1), in a manner that requires the PDGF-D C-terminal Arg residue. Stimulation with PDGF-D, but not PDGF-B, induced PDGFRβ-NRP1 complex formation in fibroblasts. Additionally, PDGF-D induced translocation of NRP1 to cell-cell junctions in endothelial cells, independently of PDGFRβ, altering the availability of NRP1 for VEGF-A-VEGFR2 signaling. PDGF-D showed differential effects on pericyte behavior in ex vivo sprouting assays compared to PDGF-B. Furthermore, PDGF-D-induced PDGFRβ-NRP1 interaction can occur in trans between molecules located in different cells (endothelial cells and pericytes). In summary, we show that NRP1 can act as a co-receptor for PDGF-D-PDGFRβ signaling and is possibly implicated in intercellular communication in the vascular wall.

The proliferation and migration of hepatic stellate cells (HSCs) profoundly impact the pathogenesis of liver inflammation and fibrogenesis. As a perennial herb native to China, Bupleurum falcatum is administered for its anti-inflammatory, antipyretic, and antihepatotoxic effects. Saikosaponin a (SSa) and Saikosaponin d (SSd) are the major active components of triterpene saponins in Bupleurum falcatum. This study analyzes how SSa and SSd affect rat HSC-T6 cell line proliferation and migration. Experimental results indicate that, in addition to suppressing HSC-T6 proliferation, wound healing activity and cell migration in a time- and dose-dependent manner, SSa and SSd significantly induce apoptosis. Additionally, SSa and SSd decreased the expressions of extracellular matrix-regulated kinase 1/2 (ERK1/2), platelet-derived growth factor receptor 1 (PDGFR1), and subsequently transforming growth factor-β1 receptor (TGF-β1R), α-smooth muscle actin, TGF-β1 and connective tissue growth factor. They also decreased phosphorylation of p38 (p-p38) and ERK1/2 (p-ERK1/2) of HSC-T6. Furthermore, both SSa and SSd can block PDGF-BB and TGF-β1-induced cell proliferation and migration of HSC-T6. These results suggest that SSa and SSd may inhibit proliferation and activation of HSC-T6, and the modulated mechanisms warrant further study.

Platelet-derived growth factor (PDGF) is thought to play a significant role in various models of vascular remodeling, particularly in the early process of vascular diseases. Its action is mediated by its specific receptor, the PDGF receptor. The PDGF alpha-receptor (PDGFalphaR) plays an important role in the growth and proliferation of vascular smooth muscle cells (VSMCs), and its gene expression is thought to be regulated by several potential transcriptional nuclear factors. However, the detailed mechanisms of tissue-specific transactivation of the PDGFalphaR gene in VSMCs remain to be clarified. We have previously demonstrated that the rat PDGFalphaR gene contains an enhancer core sequence for CCAAT/enhancer-binding proteins (C/EBPs) in its promoter region, and we have also suggested that C/EBP-delta is the principal factor involved in the induction of tissue-specific transcriptional activity of the PDGFalphaR gene in VSMCs. To explore the definitive roles of C/EBP-delta protein on PDGFalphaR gene transcription in VSMCs, we developed C/EBP-delta transgenic rats by using a chimeric fusion gene of the mouse smooth muscle alpha-actin promoter and an entire coding region of rat C/EBP-delta cDNA. This report describes the first successful targeted overexpression of C/EBP-delta capable of inducing PDGFalphaR gene transcription and modifying cell proliferative activity to PDGFs. Targeted overexpression of C/EBP-delta evokes high levels of PDGFalphaR gene expression, susceptibility to VSMC growth, and proliferation of VSMCs to PDGFs. The results obtained reveal evidence of a new role and new functional significance of C/EBP-delta on VSMC growth via the PDGFalphaR during the process of vascular remodeling and atherosclerosis.

The ability of synthetic or natural scaffolds to support invasion of cells from surrounding tissue is a key parameter for tissue engineering (TE). In this study, the migration of fibroblasts, chondrocytes, and osteoblasts into biodegradable polymer scaffolds was evaluated using a novel, three-dimensional (3-D) transmigration assay. This assay is based on a cell-populated contracted collagen lattice with a biodegradable polymer scaffold implanted at the center of the collagen gel. Cell migration into the scaffolds was assessed both quantitatively and qualitatively following various time lengths in culture using image analysis. Chondrocytes, incorporated within the collagen lattice, migrated into polymer scaffolds, when cultured both statically or in a rotating bioreactor. However, the bioreactor cultures resulted in a significantly greater cell invasion as compared to static cultures. There was a cell density-dependent osteoblast migration from collagen lattice into polymer scaffold, when tested in the transmigration assay. In addition, polymer scaffolds, treated with or without recombinant human platelet-derived growth factor (rh-PDGF-BB) were evaluated for fibroblast migration. The presence of rh-PDGF-BB resulted in significantly greater fibroblast invasion as compared to untreated scaffolds. Our studies suggest that the transmigration model provides a rapid system for testing cell invasion of potential scaffolds for tissue engineering applications.

The delta subunit of F1F0 ATPase (ATP synthase complex) is part of the stalk connecting the F1 and F0 moieties. Studies in Escherichia coli suggest that the analogous bacterial subunit, called epsilon, is essential for the ATPase assembly energy coupling. Platelet-derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS. Using two-dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the delta subunit or a closely related peptide in cultured mouse cortical neurons.

We have used a real-time quantitative RT-PCR technique (TaqMan, PE Biosystems) to identify genes that are differentially expressed by human polarised C<em>D</em>4(+) T cell subsets (Th1 or Th2). The goal was to test the feasibility of the detection method in profiling the expression of a set of marker genes important for Th1 and Th2 differentiation. We demonstrate that in polarised human Th1 cells signaling lymphocytic activation molecule (SLAM), a member of the immunoglobulin superfamily, is expressed at 7-25-fold higher levels than in Th2 cells. Along with SLAM, expression of the IL-12 receptor chain beta 2 (IL-12R beta 2) and the IFN-gamma receptor chain beta (IFN-gamma R beta) proved to be useful molecular markers indicating the state of T cell polarisation, as previously reported. Treatment with IL-12 increased SLAM mRNA expression in T cells by 3-4-fold, whereas a number of other cytokines including <em>PDGF</em>-BB, IFN-alpha A, IFN-alpha A/<em>D</em>, IFN-beta, IFN-gamma or IL-9 had no effect. Stimulating T cells by co-ligating C<em>D</em>3 and C<em>D</em>28 increased SLAM protein surface expression in both Th1 and Th2 cells. In conclusion, real-time RT-PCR detection was found to be an accurate, sensitive and highly reproducible method for fast profiling of mRNA expression in Th1 and Th2 cell subsets.