PDGF-B Signaling Research Articles

CD146⁺ Endothelial Cells Facilitate Renal Interstitial Fibrosis Through Endothelial-to-Mesenchymal Transition.

BACKGROUND Endothelial cells play a crucial role in the pathogenesis of renal interstitial fibrosis (RIF), with CD146 being upregulated on injured endothelial cells. However, the precise contribution of CD146⁺ endothelial cells to RIF remains unclear. This study aimed to observe and detect the relationship between CD146 expression and endothelial cells and to explore the role and possible mechanism of CD146 promoting endothelial-mesenchymal transition in RIF. MATERIAL AND METHODS In this study, we investigated the association between CD146⁺ endothelial cells and RIF. Double-label immunofluorescence was used in patients with chronic kidney disease, whereas multiplex immunofluorescence staining was used for the analysis in unilateral ureteral obstruction (UUO) mice. Hematoxylin and eosin and Masson trichrome staining were performed to evaluate RIF. RESULTS Our results revealed an elevation of CD146⁺ endothelial cells, which positively correlated with the degree of RIF in chronic kidney disease patients and UUO mice. Notably, CD146⁺ endothelial cells undergoing endothelial-mesenchymal transition (CD146⁺ EndMT) were significantly higher in subjects with severe renal interstitial fibrosis, as observed in chronic kidney disease patients and UUO mice. Additionally, with the progression of renal interstitial fibrosis, the expression of PDGFRb, the receptor of PDGF-B signaling pathway, increased and co-localized with CD146⁺ CD31⁺ a-SMA⁺ cells. The proportion of CD146⁺ CD31⁺ alpha-SMA⁺ PDGFRß⁺ cells in CD31⁺ cells increased. CONCLUSIONS In the process of renal interstitial fibrosis, CD146 is mainly expressed in renal interstitial vascular endothelial cells and participates in endothelial-to-mesenchymal transition, which may be related to the PDGF-B/PDGFR-ß signaling pathway.

KLF7 promotes colon adenocarcinoma progression through the PDGFB signaling pathway.

Colon adenocarcinoma (COAD) is the most common malignancy of the digestive tract, which is characterized by a dismal prognosis. No effective treatment has been established presently, thus there is an urgent need to understand the mechanisms driving COAD progression in order to develop effective therapeutic approaches and enhance clinical outcomes. In this study, we found that KLF7 is overexpressed in COAD tissues and correlated with clinicopathological features of COAD. Both gain-of-function and loss-of-function experiments have unequivocally demonstrated that overexpression of KLF7 promotes the growth and metastasis of COAD in vitro and in vivo, while KLF7 knockdown attenuated these effects. Mechanistically, our findings reveal that KLF7 can specifically bind to the promoter region of PDGFB (TGGGTGGAG), thus promoting the transcription of PDGFB and increasing its secretion. Subsequently, secreted PDGFB facilitates the progression of COAD by activating MAPK/ERK, PI3K/AKT, and JAK/STAT3 signaling pathways through PDGFRβ. Additionally, we found that sunitinib can block PDGFB signaling and inhibit COAD progression, offering a promising therapeutic strategy for COAD treatment.

PDGF-D Prodomain Differentially Inhibits the Biological Activities of PDGF-D and PDGF-B

As a member of PDGF/VEGF (Platelet-derived growth factor/ Vascular endothelial growth factor) growth factors, PDGF-D regulates blood vessel development, wound healing, innate immunity, and organogenesis. Unlike PDGF-A and PDGF-B, PDGF-D has an additional CUB (Complement C1r/C1s, Uegf, Bmp1) domain at the N-terminus of its growth factor domain, and thus it is secreted in a latent, inactive complex, which needs to be proteolytically activated for its biological activities. However, how the CUB domain contributes to the latency and activation of the growth factor remains elusive. In this study, we modeled the dimeric structure of PDGF-D pro-complex and studied the inhibitory functions of PDGF-D prodomain on PDGF-B and PDGF-D signaling. In our model, the growth factor domain of PDGF-D forms a VEGF-D-like dimer through their β1 and β3 interactions. The hinge and CUB domains of PDGF-D bind at the opposite sides of the growth factor domain and exclude the PDGFR-β (PDGF Receptor β) D2 and D3 domains from recognizing the growth factor. In addition, we verified that PDGF-D prodomain could inhibit both PDGF-B and PDGF-D mediated PDGFR-β transphosphorylation in a dose-dependent manner. However, PDGF-D prodomain could only inhibit the proliferation of NIH 3T3 cells stimulated by PDGF-D but not by PDGF-B, indicating its differential inhibitory activities toward PDGF-B and PDGF-D signaling.

Abstract MP235: PROX1 Contributes To Cardiac Valve Disease

Background: Heart valves regulate the unidirectional forward flow and prevent retrograde backflow of blood during the cardiac cycle. Cardiac valve disease (CVD) is observed in approximately 2.5% of the general population and the incidence increases to ~10% in elderly people. Patients with severe CVD require surgery and effective pharmacological treatments are currently not available. PROX1 is a transcription factor that regulates the development of lymphatic, venous, and lymphovenous valves (vascular valves). We identified that PROX1 is also expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 regulates cardiac valve development and disease is not known. Method and Results: We have discovered that mice lacking Prox1 in their VECs ( Prox1 ΔVEC ) develop enlarged aortic and mitral valves in which the expression of proteoglycans is increased (control, N=10; Prox1 ΔVEC , N=9, p <0.05). Echocardiography revealed moderate to severe stenosis of aortic valves of Prox1 ΔVEC mice (control, N=5; Prox1 ΔVEC , N=9, p <0.05). PROX1 regulates the expression of the transcription factor FOXC2 in the vascular valves. Similarly, we have found that the expression of FOXC2 is downregulated in the VECs of Prox1 ΔVEC mice. Specific knockdown of FOXC2 in VECs results in the thickening of aortic valves (control, N=10; shFoxc2 ΔVEC , N=8, p <0.05). Furthermore, restoration of FOXC2 expression in VECs ( Foxc2 OE-VEC ) ameliorates the thickening of the aortic valves of Prox1 ΔVEC mice ( Prox1 ΔVEC , N=9; Foxc2 OE-VEC ; Prox1 ΔVEC , N=8, p <0.05). We have also determined that the expression of platelet-derived growth factor-B ( Pdgfb ) is increased in the valve tissue of Prox1 ΔVEC mice and in PROX1 deficient sheep mitral valve VECs (MVECs) (siCtrl , N=4; siProx1 , N=4, p <0.05). Additionally, hyperactivation of PDGF-B signaling in mice results in a phenotype that is similar to Prox1 ΔVEC mice (control , N=4; Pdgfb GOF , N=3, p <0.05). Conclusion: Together these data suggest that PROX1 maintains the extracellular matrix composition of cardiac valves by regulating the expressions of FOXC2 and PDGF-B in VECs.

Overexpression of MiR-29b-3p Inhibits Atrial Remodeling in Rats by Targeting PDGF-B Signaling Pathway.

Purpose Studies have found that microRNAs (miRNAs) are closely associated with atrial fibrillation, but their specific mechanism remains unclear. The purpose of this experiment is to explore the function of miR-29b-3p in regulating atrial remodeling by targeting PDGF-B signaling pathway and thereby also explore the potential mechanisms. Methods We randomly divided twenty-four rats into four groups. Caudal intravenous injections of angiotensin-II (Ang-II) were administered to establish atrial fibrosis models. Expressions of miR-29b-3p and PDGF-B were then tested via RT-PCR, western blot, and immunohistochemistry. Binding sites were then analyzed via the bioinformatics online software TargetScan and verified by Luciferase Reporter. We used Masson staining to detect the degree of atrial fibrosis, while immunofluorescence and western blot were used to detect the expressions of Collagen-I and a-SMA. We used immunohistochemistry and western blot to detect the expression of connexin 43 (Cx43). Results In comparison with the Ang-II group, miR-29b-3p was seen to lower the degree of atrial fibrosis, decrease the expression of fibrosis markers such as Collagen-I and a-SMA, and increase the protein expression of Cx43. MiR-29b-3p can lower the expression of PDGF-B, while the Luciferase Reporter showed that PDGF-B is the verified target gene of miR-29b-3p. Conclusions MiR-29b-3p was able to reduce atrial structural and electrical remodeling in the study's rat fibrosis model. This biological function may be expressed through the targeted regulation of the PDGF-B signaling pathway.

Olfactomedin-like 3 promotes PDGF-dependent pericyte proliferation and migration during embryonic blood vessel formation.

Pericytes promote vessel stability and their dysfunction causes pathologies due to blood vessel leakage. Previously, we reported that Olfactomedin-like 3 (Olfml3) is a matricellular protein with proangiogenic properties. Here, we explored the role of Olfml3 in a knockout mouse model engineered to suppress this protein. The mutant mice exhibited vascular defects in pericyte coverage, suggesting that pericytes influence blood vessel formation in an Olfml3-dependent manner. Olfml3-deficient mice exhibited abnormalities in the vasculature causing partial lethality of embryos and neonates. Reduced pericyte coverage was observed at embryonic day 12.5 and persisted throughout development, resulting in perinatal death of 35% of Olfml3-deficient mice. Cultured Olfml3-deficient pericytes exhibited aberrant motility and altered pericyte association to endothelial cells. Furthermore, the proliferative response of Olfml3-/- pericytes upon PDGF-B stimulation was significantly diminished. Subsequent experiments revealed that intact PDGF-B signaling, mediated via Olfml3 binding, is required for pericyte proliferation and activation of downstream kinase pathways. Our findings suggest a model wherein pericyte recruitment to endothelial cells requires Olfml3 to provide early instructive cue and retain PDGF-B along newly formed vessels to achieve optimal angiogenesis.

Knockout of EGFL6 by CRISPR/Cas9 Mediated Inhibition of Tumor Angiogenesis in Ovarian Cancer.

Tumor angiogenesis plays an important role in the progression and metastasis of ovarian cancer. EGFL6 protein is highly expressed in ovarian cancer and has been proposed to play an important role in promoting tumor angiogenesis. Here, a CRISPR/Cas9 system was used to knockout the EGFL6 gene in the ovarian cancer cell line SKOV3, using specific guide RNA targeting the exons of EGFL6. The knockout of EGFL6 markedly inhibited the proliferation, migration, and invasion of SKOV3 cells, as well as promoted apoptosis of tumor cells. In the nude mouse model of ovarian cancer, knockout of EGFL6 remarkably inhibited tumor growth and angiogenesis. The transcript profile assays detected 4,220 differentially expressed genes in the knockout cells, including 87 genes that were correlated to proliferation, migration, invasion, and angiogenesis. Moreover, Western blotting confirmed that EGFL6 knockout downregulated the FGF-2/PDGFB signaling pathway. Thus, the results of this study indicated that EGFL6 could regulate cell proliferation, migration, and angiogenesis in ovarian cancer cells by regulating the FGF-2/PDGFB signaling pathway.

PDGF-B: The missing piece in the mosaic of PDGF family role in craniofacial development.

The platelet-derived growth factor (PDGF) family consists of four ligands (PDGF-A, PDGF-B, PDGF-C, PDGF-D) and two tyrosine kinase receptors (PDGFR-α and PDGFR-β). In vertebrates, PDGF signaling influences cell proliferation, migration, and matrix deposition, and its up-regulation is implicated in cancer progression. Despite this evidence, the role of each family member during embryogenesis is still incomplete and partially controversial. In particular, study of the role of pdgf signaling during craniofacial development has been focused on pdgf-a, while the role of pdgf-b is almost unknown due to the lethal phenotypes of pdgf-b-null mice. By using a pdgf-b splice-blocking morpholino approach, we highlighted impairment of neural crest cell (NCC) migration in Xenopus laevis morphants, leading to alteration of NCC derivatives formation, such as cranial nerves and cartilages. We also uncovered a possible link between pdgf-b and the expression of cadherin superfamily members cdh6 and cdh11, which mediate cell-cell adhesion promoting NCC migration. Our results suggested that pdgf-b signaling is involved in cranial NCC migration and it is required for proper formation of craniofacial NCC derivatives. Taken together, these data unveiled a new role for pdgf-b during vertebrate development, contributing to complete the picture of pdgf signaling role in craniofacial development.

Lung Hydrogel Implantation System to Study the Pathogenesis of Pulmonary Hypertension

Remodeling of distal pulmonary arterioles (PAs) associated with marked accumulation of pulmonary artery smooth muscle cells (PASMCs) represents one of the major pathologic features of pulmonary hypertension (PH). The levels of a transcription factor, Twist1 are upregulated in the lungs of patients with pulmonary arterial hypertension (PAH). However, the mechanism by which endothelial Twist1 stimulates SMC accumulation to distal PAs in PH remains unclear. Here we have found that Twist1 overexpression increases the expression of platelet‐derived growth factor (PDGFB) in cultured human pulmonary arterial endothelial cells (HPAECs). Conditioned media collected from Twist1 overexpressing HPAECs induce SMC migration and growth. Knockdown of Twist1 in ECs attenuates hypoxia‐induced accumulation of alpha‐smooth muscle actin (SMA)‐positive cells to the PAs in mice. To further study the mechanism by which endothelial Twist1 controls vascular remodeling and SMC accumulation in the lung, we implant fibrin gel supplemented with fluorescently labeled HPAECs on the mouse lung. Supplemented HPAECs form vascular structures and hypoxia treatment stimulates accumulation of alpha‐SMA–positive cells to blood vessels and upregulates expression of PDGFB in the gel. Twist1 overexpression in supplemented HPAECs or hypoxia treatment also induces endothelial‐to‐mesenchymal transition (EndMT) in the implanted gel, which also partly contributes to the pathogenesis of PH. Endothelial Twist1 plays a key role in SMC accumulation to PAs through PDGFB signaling and the mouse lung gel implantation system would be a good model system to study the pathogenesis of PH and other lung diseases.Support or Funding InformationAHA18TPA34170129, MCW Research Affair Committee New Investigator AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantitative proteomic analysis of gastric cancer tissue reveals novel proteins in platelet-derived growth factor b signaling pathway.

Gastric cancer is one of the most common cancers in Asian countries. Searching for reliable biomarkers involving the development of gastric cancer is important for clinical practice. Quantitative proteomics has become an important method contributed to the discovery of novel diagnostic or therapeutic targets for the management of cancer. Here, we identified differently expressed proteins in gastric cancer and normal gastric tissues by using the high resolution mass spectrometer. Among the total of 2280 identified proteins, 87 were differentially expressed between gastric cancer and normal gastric tissues. Notably, several significant proteins are in the PDGF-B signaling pathway, including peroxiredoxin5 (PRDX5), S100A6, calreticulin (CALR) and cathepsin D (CTSD), which were validated by western blot. Furthermore, upstream regulators including PDGF-B, PDGFR-β, Akt, eIF4E and p70s6K were found significantly increased in the gastric cancer tissues. In addition, silencing of PRDX5 and PDGF-B suppressed the proliferation of gastric cancer cells in vitro. The administration of exogenous PDGF-BB recovered the reduced expression of PDGF-B signaling pathway in PDGF-B knockdown cells. Taken together, our findings suggested that PDGF-B signaling pathway plays an important role in the regulation of gastric cancer proliferation and the inhibition of this pathway may be a potential approach for treatment of gastric cancer.

Pre-Clinical Study of Panobinostat in Xenograft and Genetically Engineered Murine Diffuse Intrinsic Pontine Glioma Models.

BackgroundDiffuse intrinsic pontine glioma (DIPG), or high-grade brainstem glioma (BSG), is one of the major causes of brain tumor-related deaths in children. Its prognosis has remained poor despite numerous efforts to improve survival. Panobinostat, a histone deacetylase inhibitor, is a targeted agent that has recently shown pre-clinical efficacy and entered a phase I clinical trial for the treatment of children with recurrent or progressive DIPG.MethodsA collaborative pre-clinical study was conducted using both a genetic BSG mouse model driven by PDGF-B signaling, p53 loss, and ectopic H3.3-K27M or H3.3-WT expression and an H3.3-K27M orthotopic DIPG xenograft model to confirm and extend previously published findings regarding the efficacy of panobinostat in vitro and in vivo.ResultsIn vitro, panobinostat potently inhibited cell proliferation, viability, and clonogenicity and induced apoptosis of human and murine DIPG cells. In vivo analyses of tissue after short-term systemic administration of panobinostat to genetically engineered tumor-bearing mice indicated that the drug reached brainstem tumor tissue to a greater extent than normal brain tissue, reduced proliferation of tumor cells and increased levels of H3 acetylation, demonstrating target inhibition. Extended consecutive daily treatment of both genetic and orthotopic xenograft models with 10 or 20 mg/kg panobinostat consistently led to significant toxicity. Reduced, well-tolerated doses of panobinostat, however, did not prolong overall survival compared to vehicle-treated mice.ConclusionOur collaborative pre-clinical study confirms that panobinostat is an effective targeted agent against DIPG human and murine tumor cells in vitro and in short-term in vivo efficacy studies in mice but does not significantly impact survival of mice bearing H3.3-K27M-mutant tumors. We suggest this may be due to toxicity associated with systemic administration of panobinostat that necessitated dose de-escalation.

Inhibition of Glomerular Mesangial Cell Proliferation by siPDGF-B- and siPDGFR-β-Containing Chitosan Nanoplexes.

Mesangioproliferative glomerulonephritis is a disease that has a high incidence in humans. In this disease, the proliferation of glomerular mesangial cells and the production of extracellular matrix are important. In recent years, the RNAi technology has been widely used in the treatment of various diseases due to its capability to inhibit the gene expression with high specificity and targeting. The objective of this study was to decrease mesangial cell proliferation by knocking down PDGF-B and its receptor, PDGFR-β. To be able to use small interfering RNAs (siRNAs) in the treatment of this disease successfully, it is necessary to develop appropriate delivery systems. Chitosan, which is a biopolymer, is used as a siRNA delivery system in kidney drug targeting. In order to deliver siRNA molecules targeted at PDGF-B and PDGFR-β, chitosan/siRNA nanoplexes were prepared. The in vitro characterization, transfection studies, and knockdown efficiencies were studied in immortalized and primary rat mesangial cells. In addition, the effects of chitosan nanoplexes on mesangial cell proliferation and migration were investigated. After in vitro transfection, the PDGF-B and PDGFR-β gene silencing efficiencies of PDGF-B and PDGFR-β targeting siRNA-containing chitosan nanoplexes were 74 and 71% in immortalized rat mesangial cells and 66 and 62% in primary rat mesangial cells, respectively. siPDGF-B- and siPDGFR-β-containing nanoplexes indicated a significant decrease in mesangial cell migration and proliferation. These results suggested that mesangial cell proliferation may be inhibited by silencing of the PDGF-B signaling pathway. Gene silencing approaches with chitosan-based gene delivery systems have promise for the efficient treatment of renal disease.

Platelet-derived Growth Factor-B Protects Rat Cardiac Allografts From Ischemia-reperfusion Injury.

Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-β, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.

Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells.

To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling. The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-β1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid β-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor β (PDGFRβ) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRβ and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid β-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls (P < 0.01). Cells containing the cystine knot of BMP-4 expressed the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-β1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of β-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRβ receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRβ and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2. CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-β. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

TGFβ and PDGF-B signaling blockade inhibits myofibroblast development from both bone marrow-derived and keratocyte-derived precursor cells in vivo

Myofibroblasts, the primary cells associated with corneal stromal haze (opacity), can be derived from both cornea-derived and bone marrow-derived precursor cells. In the present study, the role of TGFβ or PDGF blockage on bone marrow-derived myofibroblast development was investigated using a green fluorescent protein (GFP) chimeric bone marrow mouse model and plasmid vectors that blocked TGFβ or PDGF signaling. At the peak of corneal haze one month after irregular phototherapeutic keratectomy the central stroma had significantly less alpha-smooth muscle actin (α-SMA)-positive cells derived from GFP+ bone marrow-derived cells or GFP− keratocyte/corneal fibroblast-derived cells when corneas were treated with the TGFβ blocking vector pGFPC1.TGFRBKDEL or the PDGF blocking vector pCMV.PDGFRB.23KDEL compared with the corresponding empty vector treated or untreated control groups. In individual animals, 30–60% of myofibroblasts were derived from bone marrow-derived precursor cells and 40–70% of myofibroblasts were derived from keratocyte-derived precursor cells. TGFβ and PDGF regulate corneal myofibroblast development from bone marrow-derived precursor cells and keratocyte/corneal fibroblast-derived precursor cells.

LIM-homeobox gene 2 promotes tumor growth and metastasis by inducing autocrine and paracrine PDGF-B signaling

An epithelial-mesenchymal transition (EMT) is a critical process during embryonic development and the progression of epithelial tumors to metastatic cancers. Gene expression profiling has uncovered the transcription factor LIM homeobox gene 2 (Lhx2) with up-regulated expression during TGFβ-induced EMT in normal and cancerous breast epithelial cells. Loss and gain of function experiments in transgenic mouse models of breast cancer and of insulinoma in vivo and in breast cancer cells in vitro indicate that Lhx2 plays a critical role in primary tumor growth and metastasis. Notably, the transgenic expression of Lhx2 during breast carcinogenesis promotes vessel maturation, primary tumor growth, tumor cell intravasation and metastasis by directly inducing the expression of platelet-derived growth factor (PDGF)-B in tumor cells and by indirectly increasing the expression of PDGF receptor-β (PDGFRβ) on tumor cells and pericytes. Pharmacological inhibition of PDGF-B/PDGFRβ signaling reduces vessel functionality and tumor growth and Lhx2-induced cell migration and cell invasion. The data indicate a dual role of Lhx2 during EMT and tumor progression: by inducing the expression of PDGF-B, Lhx2 provokes an autocrine PDGF-B/PDGFRβ loop required for cell migration, invasion and metastatic dissemination and paracrine PDGF-B/PDGFRβ signaling to support blood vessel functionality and, thus, primary tumor growth.

Platelet-Derived Growth Factor (PDGF)-C Neutralization Reveals Differential Roles of PDGF Receptors in Liver and Kidney Fibrosis

Platelet-derived growth factors (PDGF) are key mediators of organ fibrosis. We investigated whether PDGF-C(-/-) mice or mice treated with neutralizing PDGF-C antibodies are protected from bile duct ligation-induced liver fibrosis, and we compared the effects with those of PDGF-C deficiency or neutralization on kidney fibrosis induced by unilateral ureteral obstruction. Unexpectedly, and in contrast to kidney fibrosis, PDGF-C deficiency or antagonism did not protect from liver fibrosis or functional liver impairment. Furthermore, the hepatic infiltration of monocytes/macrophages/dendritic cells and chemokine mRNA expression (CC chemokine ligand [CCL]5, CCL2, and CC chemokine receptor 2 [CCR2]) remained unchanged. Transcript expression of PDGF ligands increased in both liver and kidney fibrosis and was not affected by neutralization of PDGF-C. In kidney fibrosis, PDGF-C deficiency or antagonism led to reduced expression and signaling of PDGF-receptor (R)-α- and PDGFR-β-chains. In contrast, in liver fibrosis there was either no difference (PDGF-C(-/-) mice) or even an upregulation of PDGFR-β and signaling (anti-PDGF-C group). Finally, invitro studies in portal myofibroblasts pointed to a predominant role of PDGF-B and PDGF-D signaling in liver fibrosis. In conclusion, our study revealed significant differences between kidney and liver fibrosis in that PDGF-C mediates kidney fibrosis, whereas antagonism of PDGF-C in liver fibrosis appears to be counteracted by significant upregulation and increased PDGFR-β signaling. PDGF-C antagonism, therefore, may not be effective to treat liver fibrosis.

PDGFRβ expression in tumor stroma of pancreatic adenocarcinoma as a reliable prognostic marker

Pancreatic adenocarcinoma is a lethal disease that often develops a desmoplastic reaction in tumor stroma. In general, desmoplasia is thought to promote tumor growth. However, its molecular pathology and prognostic potential have not been fully investigated. Here, we investigate 26 cases of pancreatic ductal adenocarcinoma and examine the clinicopathological association between survival and expression levels of several molecular markers for stromal cells. These include alpha-smooth muscle actin (SMA) and platelet-derived growth factor (PDGF) receptor β (PDGFRβ). Both are markers of activated fibroblasts or pancreatic stellate cells (PSCs) that play an important role in desmoplasia. The staining patterns of both molecular markers were not uniform, so we categorized them into 3 grades (high, middle, and low) according to intensity. Interestingly, Kaplan-Meier analysis revealed that higher expression of PDGFRβ matched shorter prognosis (p=0.0287, log-rank test) as well as lymphatic invasion and lymph node metastasis, whereas SMA did not (p=0.6122). Our results suggest the prognostic potential of cancer stroma via PDGF-B signaling. Regulation of PDGF-B-associated signaling crosstalk between cancer cells and stroma cells, therefore, may indicate a possible therapeutic target for desmoplastic malignant tumors such as pancreatic adenocarcinoma.

Trophoblasts Regulate the Placental Hematopoietic Niche through PDGF-B Signaling

The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRβ, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.

Abstract 114: Neuroprotective Role of Brain Pericytes through PDGFRβ-Akt Signaling in Ischemic Stroke

Brain pericytes are a constituent of the neurovascular unit and play various important roles in brain functions, such as regulation of capillary blood flow, maintenance of blood-brain barrier and angiogenesis. Previous reports have elucidated that PDGF-B prevents neuronal cell death during ischemic insults in adult rodent models; however, the detailed mechanisms by which PDGF-B signaling protects neurons from ischemic damage are not fully understood. In the present study, we investigated whether brain pericytes play neuroprotective roles in brain ischemia, using a permanent middle cerebral artery occlusion stroke model (MCAO) and cultured human brain pericytes. Immunohistochemistry revealed that the expression of PDGF receptorβ(PDGFRβ) was induced predominantly in pericytes in peri-infarct areas. PDGF-B induced marked phosphorylation of Akt in cultured pericytes. Consistently, Akt was markedly phosphorylated in the PDGFRβ-expressing pericytes in peri-infarct areas. PDGF-B upregulated the expression of neurotrophins, such as neuronal growth factor (NGF) and neurotrophin-3 (NT-3), through Akt activation in the cultured pericytes. We subjected PDGFRβheterozygous knockout (PDGFRβ+/-) mice to MCAO. Infarct volume, as assessed by MAP2 immunostaining, was significantly greater in PDGFRβ+/- than wild-type mice ( 48% increase at day 7, p &lt; 0.01 , n=5). The number of TUNEL positive apoptotic cells was significantly greater in PDGFRβ+/- mice (54 % increase at day 4, p &lt; 0.001 , n=6). Production of NGF and NT-3 at mRNA and protein levels in infarct areas was significantly decreased in PDGFRβ+/- mice (NGF: 28% decrease, p&lt;0.05, NT-3: 22% decrease, p&lt;0.05). Since it has been established that neurotrophin receptors are induced in peri-infarct areas, the decreases in neurotrophin production may increase apoptotic neuronal cell death in the PDGFRβ+/- mice. In conclusion, brain pericytes may have a direct neuroprotective role through secreting neurotrophins via PDGFRβ-Akt signaling, thereby decreasing infarct volume in ischemic stroke.