PDGF-B Expression Research Articles

Abstract PD9-11: Platelet derived growth factor receptor-β signaling: A novel therapeutic target for breast cancer associated brain metastasis

Abstract PDGFRβ is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand, PDGFB, is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stromal signaling mediates breast cancer initiation, progression, and metastasis remains unclear. To test this important research question, we developed a mouse model of mesenchymal-specific PDGFRβ hyper-activation. PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium during normal development. To test the effect of hyper-active mesenchymal PDGFRβ on disease progression, experimental tail vein metastasis assays were performed where we observed prominent brain metastases in 50% of the PDGFRβ mutantmice (n=5/10) with no brain lesions seen in controls (n=0/19). There was no difference in the incidence of lung or liver metastases in the mutant mice suggesting a pro-metastatic function for PDGFRβ in the brain metastatic niche. To rule out dysfunction of the blood brain barrier contributing to the observed metastatic spread, we then intracranially injected mammary tumor cells, and as expected based on our metastasis assay, found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, these combined findings are the first example where genetic manipulation of the stroma increases breast cancer associated brain metastases (BCBM). Given that these pre-clinical data suggest that primary breast tumors expressing high PDGFB could preferentially metastasize to the brain, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors (total n=425). While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. These findings further suggest that high primary tumor PDGFBexpression defines a subset of breast cancer patients predisposed to brain metastases and that these patients may benefit from therapeutic inhibition of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR specific inhibitor crenolanib. Excitingly, crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings to date (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM. Ongoing studies are evaluating how the PDGFRβ-expressing mesenchymal cells within the brain promote a pro-metastatic niche. Citation Format: Sizemore GM, Hammer AM, Thies KA, Hildreth BE, Russell LO, Sizemore ST, Trimboli AJ, Kladney RD, Steck SA, Das M, Bolyard CM, Pilarski R, Schoenfield L, Otero J, Chakravarti A, Ringel M, Kaur B, Leone G, Ostrowski MC. Platelet derived growth factor receptor-β signaling: A novel therapeutic target for breast cancer associated brain metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-11.

Novel insights into the evolution of the caveolin superfamily and mechanisms of antiapoptotic effects and cell proliferation in lamprey

Caveolin-1 is the main structural and functional component of caveolin, and it is involved in the regulation of cholesterol transport, endocytosis, and signal transduction. Moreover, changes in caveolin-1 play an important role in tumorigenesis and inflammatory processes. Previous studies have demonstrated that human caveolin-1 is mainly located in the cell membrane and exhibits cell type- and stage-dependent functional differences during cancer development and inflammatory responses. However, the role of Lamprey-caveolin-like (L-caveolin-like) in lamprey remained unknown. Here, we demonstrated that L-caveolin-like performs anti-inflammation and oncogenic functions and the function of caveolin-1 diverged during vertebrate evolution. Moreover, the results reveal the mechanism underlying the antiapoptotic effects of L-caveolin-like. An L-caveolin-like gene from Lampetra japonica (L. japonica) was identified and characterized. L-Caveolin-like was primarily distributed in the leukocytes, intestines and supraneural bodies (Sp-bodies) immune organs as indicated by Q-PCR and immunohistochemistry assays. The mRNA and protein expression levels of L-caveolin exhibited consistent increases in expression at 2 and 72 h in adult tissues after exposure to lipopolysaccharide (LPS) and in leukocytes stimulated by Vibrio anguillarum (V. anguillarum), Staphylococcus aureus (S. aureus), and Poly I:C. Furthermore, the overexpression of pEGFP-N1-L-caveolin-like was associated with a distinct localization in mitochondria, with decreased cytochrome C (Cyt C) and mitochondrial Cyt C oxidase subunit I (CO I) expression. In addition, increased cellular ATP levels suggested that this protein prevented mitochondrial damage. The overexpression of pEGFP-N1-L-caveolin-like led to the altered expression of factors related to apoptosis, such as decreased Caspase-9, Caspase-3, p53, and Bax expression and increased Bcl-2 expression. In addition, the overexpression of pEGFP-N1-L-caveolin-like promoted cell proliferation associated with upregulated EGF, bFGF, and PDGFB expression. Together, these findings indicated that the L-caveolin-like protein from L. japonica induced the activation of antiapoptotic effects via the mitochondrial Cyt C-mediated Caspase-3 signaling pathway. Our analysis further suggests that L-caveolin-like is an oncogene protein product and anti-inflammatory molecule from lamprey that evolved early in vertebrate evolution.

XPD suppresses cell proliferation and migration via miR-29a-3p-Mdm2/PDGF-B axis in HCC

ObjectiveThe aim of this study was to investigate the role of XPD in migration and invasion of hepatocellular carcinoma (HCC) cells.MethodsThe expression of XPD and miR-29a-3p was examined by western blot and qRT-PCR, cell proliferation was detected by MTT assay, cell migration was detected by transwell assay. TargetScan was used to predict potential targets of miR-29a-3p.ResultsIn this study, we found that the expression of XPD and miR-29a-3p was downregulated in HCC samples and HCC cell lines. XPD suppressed proliferation and migration of HCC cell via regulating miR-29a-3p expression. Target prediction analysis and dual-luciferase reporter assay confirmed Mdm2 and PDGF-B were direct targets of miR-29a-3p, and miR-29a-3p suppressed proliferation and migration of HCC cells via regulating the expression of Mdm2 or PDGF-B.ConclusionsOur data indicated that XPD suppressed cell proliferation and migration via miR-29a-3p-Mdm2/PDGF-B axis in HCC.

Impaired adrenergic agonist-dependent beige adipocyte induction in obese mice.

Brown adipocytes, which exist in brown adipose tissue (BAT), are activated by adrenergic stimulation, depending on the activity of uncoupling protein 1 (UCP1). Beige adipocytes emerge from white adipose tissue (WAT) in response to chronic adrenergic stimulation. We investigated obesity-related changes in responses of both types of adipocytes to adrenergic stimulation in mice. Feeding of mice with high-fat diets (HFD: 45%-kcal fat) for 14 weeks resulted in significantly higher body and WAT weight compared to feeding with normal diets (ND: 10%-kcal fat). Injection with β3-adrenergic receptor agonist CL316,243 (CL; 0.1 mg/kg, once a day) for one week elevated the mRNA and protein expression levels of UCP1 in BAT, irrespective of diet. In WAT, CL-induced UCP1 expression in ND mice; however, the responses to CL treatment were attenuated in HFD mice, indicating that CL-induced browning of WAT was impaired in obese mice. Flow cytometric analysis revealed a significant decrease in platelet-derived growth factor receptor (PDGFR) α-expressing beige adipocyte progenitors in WAT of HFD mice compared with those of ND mice. Expression of PDGF-B, a PDGFRα ligand, increased in WAT following CL-injection in ND mice, but not in HFD mice. Treatment of mice with a PDGFR inhibitor significantly decreased CL-dependent UCP1 protein induction in WAT. Our study demonstrates that β3-adrenergic stimulation-dependent beige adipocyte induction in WAT is impaired by obesity in mice, potentially due to obesity-dependent reduction in the number of PDGFRα-expressing progenitors and decreased PDGF-B expression.

Unraveling reno-protective effects of SGLT2 inhibition in human proximal tubular cells.

Large clinical trials demonstrated that SGLT2 inhibitors (SGLT2i) slow the progression of kidney function decline in type 2 diabetes. Because the underlying molecular mechanisms are largely unknown, we studied the effects of SGLT2i on gene expression in two human proximal tubular (PT) cell lines under normoglycemic conditions, utilizing two SGLT2i, namely empagliflocin and canagliflocin. Genome-wide expression analysis did not reveal substantial differences between these two SGLT2i. Microarray hybridization analysis identified 94 genes that were both upregulated by TGF-β1 and downregulated by either of the two SGLT2i in HK-2 and RPTEC/TERT1 (renal proximal tubular epithelial cells/telomerase reverse transcriptase 1) cells. Extracellular matrix organization showed the highest significance in pathway enrichment analysis. Differential gene expression of three annotated genes of interest within this pathway was verified on mRNA level in both cell lines. Whereas TGF-β1 induced mRNA expression of thrombospondin 1 (THBS1; 4.3-fold), tenascin C (TNC; 8-fold), and platelet-derived growth factor subunit B (PDGF-B; 4.2-fold), SGLT2i downregulated basal mRNA expression of THBS1 (0.2-fold), TNC (0.5 fold), and PDGF-B (0.6-fold). Administration of SGLT2i in the presence of TGF-β1 resulted in a significant inhibition of TGF-β1-induced THBS1 and TNC mRNA expression and TGF-β1-induced THBS1, TNC, and PDGF-BB protein expression. We conclude that SGLT2i block basal and TGF-β1-induced expression of key mediators of renal fibrosis and kidney disease progression in two independent human PT cell lines.

Expression of platelet-derived growth factor B is upregulated in patients with thoracic aortic dissection

ObjectiveThoracic aortic dissection (TAD) is a serious condition requiring urgent treatment to avoid catastrophic consequences. The inflammatory response is involved in the occurrence and development of TAD, possibly potentiated by platelet-derived growth factors (PDGFs). This study aimed to determine whether expression of PDGF-B (a subunit of PDGF-BB) was increased in TAD patients and to explore the factors responsible for its upregulation and subsequent effects on TAD. MethodsFull-thickness ascending aorta wall specimens from TAD patients (n = 15) and control patients (n = 10) were examined for expression of PDGF-B and its receptor (PDGFRB) and in terms of morphology, inflammation, and fibrosis. Blood samples from TAD and control patients were collected to detect plasma levels of PDGF-BB and soluble elastins. ResultsExpression levels of PDGF-B, PDGFRB, and collagen I were significantly enhanced in ascending aorta wall specimens from TAD patients compared with controls. Furthermore, soluble elastic fragments and PDGF-BB were significantly increased in plasma from TAD patients compared with controls, and numerous irregular elastic fibers and macrophages were seen in the ascending aorta wall in TAD patients. ConclusionsAn increase in elastic fragments in the aorta wall might be responsible for inducing the activation and migration of macrophages to injured sites, leading to elevated expression of PDGF-B, which in turn induces deposition of collagen, disrupts extracellular matrix homeostasis, and increases the stiffness of the aorta wall, resulting in compromised aorta compliance.

Radiation-Induced Pulmonary Fibrosis (RIPF) Is Mediated By Interleukin 13 Receptor Subunit Alpha 2 (IL13Ra2)

Radiation induced pulmonary fibrosis (RIPF) is a late toxicity of thoracic radiotherapy. Type 2 immune responses driven by IL-13 have recently been implicated in the pathogenesis of RIPF, in part through interaction with the heterodimeric IL-13Ra1/IL4Rα receptor complex. Although its mechanism is not clearly understood, IL-13Rα2 is a transmembrane receptor thought to function as a decoy receptor for IL-13. In this study, we sought to characterize the role of IL13Rα2. Recent evidence implicates IL-13Rα2 in signaling of chitinases produced in type 2 inflammatory responses, which play a role in RIPF initiation and progression. The 10 week old C57BL6/NcR (WT) and IL13Ra2 deficient [C57BL/6-[Ko]IL13Rα2 (IL-13Rα2-/-)] mice were exposed to 5 daily 6 Gy fractions of thoracic radiation (RT). Lung tissue was collected at 16 weeks after RT. Collagen accumulation was measured with hydroxyproline content and accumulation of total and alternatively activated (M2) macrophages was assessed with immunohistochemistry. Bone marrow derived macrophages (BMDM) from each strain were treated with IL-13. Gene expression in RNA isolated from lung tissue and BMDM was evaluated with quantitative RT-PCR and PCR array. Deficiency of IL13Rα2 significantly extended survival significantly after thoracic irradiation compared to WT mice (19 vs. 21 weeks, p=0.03 log rank). WT mice developed confluence of fibrosis, increased hydroxyproline content, and accumulation of M2 macrophages after RT. IL-13Ra2-/- mice had no observable increase in hydroxyproline content after RT and had no evidence of macrophage accumulation when compared to unirradated controls. The expression of fibrosis-associated cytokines and chemokines (Ccl2, Timp1, and IL-6) was suppressed in irradiated IL-13Ra2-/-lung compared to WT. WT macrophages isolated from whole lung expressed increased levels both soluble and membrane bound isoforms of IL13Rα2 after radiation. BMDM from IL-13Ra2-/- mice treated with IL-13 had suppressed expression of Ccl11, Timp1, and Pdgfb compared to BMDM from WT mice. Our data show that IL-13Rα2 deficiency attenuates RIPF, and that this protection is accompanied by decreased accumulation of macrophages and altered production of inflammatory cytokines and chemokines by macrophages. Whether the protective benefit of IL-13Rα2 deficiency is attributable to absent decoy-receptor function is currently being investigated.

Essential role of suppressor of cytokine signaling 1 (SOCS1) in hepatocytes and macrophages in the regulation of liver fibrosis

The hepatic fibrogenic response is a protective mechanism activated by hepatocyte damage and is resolved upon elimination of the cause. However, persistent injuries cause liver fibrosis (LF) to evolve into cirrhosis, which promotes the development of hepatocellular carcinoma (HCC). Development of efficient treatments for LF requires better understanding the underlying molecular pathogenic mechanisms. The loss of suppressor of cytokine signaling 1 (SOCS1) expression promotes LF and HCC in human and mice, but the underlying mechanisms remain unclear. SOCS1 is a key regulator of immune cell activation. To investigate the anti-fibrogenic functions of SOCS1 in hepatocytes and macrophages, we generated mice lacking SOCS1 in hepatocytes (Socs1fl/flAlbCre) or macrophages (Socs1fl/flLysMCre) and evaluated hepatic fibrogenic response to carbon tetrachloride (CCl4). Socs1fl/flAlbCre and Socs1fl/flLysMCre mice showed severe LF characterized by increased collagen deposition, hydroxyproline content, myofibroblast accumulation along with elevated expression of Acta2 and Col1a1 genes. CCl4 treatment triggered significant damage to hepatocytes in Socs1fl/flAlbCre mice but not in Socs1fl/flLysMCre mice. In both mice CCl4 treatment reduced the expression of Mmp2 and increased the expression of Timp1. SOCS1 deficiency in hepatocytes or macrophages did not affect Il6, Tnfa or Tgfb, but diminished Infg and augmented Pdgfb expression. Both Socs1fl/flAlbCre and Socs1fl/flLysMCre livers showed increased mononuclear cell infiltration accompanied by elevated Ccl2 expression. Our findings show that SOCS1 exerts non-redundant functions in hepatocytes and macrophages to regulate the hepatic fibrogenic response possibly through limiting hepatocyte damage and the inflammatory response of macrophages, and support the idea of exploiting SOCS1 in LF treatment.

Abstract 49: Stromal platelet derived growth factor receptor (PDGFRβ) signaling: A novel therapeutic target for breast cancer brain metastasis (BCBM)

Abstract Stromal PDGFRβ has emerged as an actionable mediator of breast tumor-stromal communication. As a receptor tyrosine kinase, PDGFRβ is activated by its ligand, PDGFB, which is released by neighboring tumor epithelium and endothelium. However, how PDGF signaling mediates breast cancer initiation, progression, and metastasis remains unclear. To evaluate PDGFRβ in this disease, we developed a mouse model of stromal-specific PDGFRβ activation using the Fsp-cre transgene previously published by our group (PDGFRβ mutant). PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium. To evaluate the functional relevance of PDGFRβ activation on metastatic progression, we performed tail vein injection of PDGFB expressing murine mammary tumor cells and, surprisingly, observed brain metastases in 50% of the PDGFRβ mutant mice while no brain lesions were seen in controls. There was no difference in the incidence of lung, liver or bone metastases. Mammary tumor cells expressing low PDGFB did not exhibit a similar increase in brain metastases in mutant mice. While there is no observable difference in blood brain barrier permeability in the mutant mice, we bypassed this variable by intracranially injecting mammary tumor cells and found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of BCBM. Also, our pre-clinical data suggests that primary breast tumors that express high PDGFB could preferentially metastasize to the brain. To test this in patients, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors. While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. Our findings suggest high primary tumor PDGFB expression defines a subset of breast cancer patients predisposed to brain metastases. These patients may benefit from therapeutic intervention of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR specific inhibitor, Crenolanib. Excitingly, Crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM. Citation Format: Katie A. Thies, Anisha M. Hammer, B. Eason Hildreth, Luke O. Russell, Steven T. Sizemore, Anthony J. Trimboli, Raleigh D. Kladney, Chelsea M. Bolyard, Robert Pilarski, Lynn Schoenfield, Jose Otero, Arnab Chakravarti, Matthew Ringel, Balveen Kaur, Gustavo Leone, Michael C. Ostrowski, Gina M. Sizemore. Stromal platelet derived growth factor receptor (PDGFRβ) signaling: A novel therapeutic target for breast cancer brain metastasis (BCBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 49.

Blockade of Tyrosine Kinases as Preventive Strategy Against Cardiac Allograft Vasculopathy in a Murine Aortic Transplant Model

Objectives Previous reports suggest a role of platelet derived growth factor (PDGF) in the development of cardiac allograft vasculopathy (CAV). The pharmaceutical blockade of tyrosine kinases may alter the expression of different growth factor receptors: platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) and hereby play a role in the context of cardiac allograft vasculopathy. Therefore the aim of this study was to determine the impact of tyrosine kinase inhibition on the development of cardiac allograft vasculopathy in a mouse model. Methods Fully allogeneic mouse aortas from C57BL/6 mice (H2b) were abdominally transplanted into CBA mice (H2k). Recipients were treated with tyrosine kinase inhibitor nintedanib (60mg/kg/d) and sacrificed on day 14 for cytokine PCR analysis of the graft or on day 30 for (immuno-) histological measurements. Results Aortic grafts treated with tyrosine kinase inhibitor nintedanib showed significantly reduced neointima proliferation (33% ± 12% vs. 54% ± 13%) after 30 days. These findings go along with significantly reduced amounts of smooth muscle cells (SMC) (20% ± 9% vs. 42% ± 10%) within the neointima. Further immunofluorescence analysis revealed no relevant difference in the percentages of dendritic cells, macrophages and CD4+ T-cells within the neointima. Meanwhile, the expression of both PDGF receptor subtypes α and β was reduced within the neointima of the aortic grafts. Additionally, we found a reduced gene expression of the ligand PDGF-B, a mitogen for SMC, and of the pro-inflammatory CD40L. Conclusion PDGF receptor blockade via inhibition of receptor tyrosine kinases seems to be a promising way to prevent the development of cardiac allograft vasculopathy in a mouse model. We would speculate a direct inhibition of the migration and proliferation of smooth muscle cells with a resulting decrease of neointima formation as the mechanism of action.

Nanosphere-mediated co-delivery of VEGF-A and PDGF-B genes for accelerating diabetic foot ulcers healing in rats.

Diabetic ischemic ulcer is an intractable diabetic complication. Angiogenesis is a critical factor for wound healing in patients with diabetic foot wounds. Sustained gene delivery could be notably necessary in modulating gene expression in chronic ulcer healing and might be a promising approach for diabetic foot ulcers. In the present study, Sprague-Dawley rats were used to establish diabetic foot ulcer models by streptozotocin and skin biopsy punch. The plasmids expressing VEGF-A and PDGF-B were prepared and then incorporated with polylactic-co-glycolic acid (PLGA) nanospheres to upregulate genes expression. The aim of this study was to explore whether the engineered VEGF-A and PDGF-B based plasmid-loaded nanospheres could be upregulated in streptozotocin-induced diabetic rats and improve the wound healing. The cultured fibroblasts could be effectively transfected by means of nanosphere/plasmid in vitro. In vivo, the expression of VEGF-A and PDGF-B was significantly upregulated at full-thickness foot dorsal skin wounds and the area of ulceration was progressively and significantly reduced following treatment with nanosphere/plasmid. These results indicated that combined gene transfer of VEGF-A and PDGF-B could improve reparative processes in the wounded skin of diabetic rats and nanosphere may be a potential non-viral vector for gene therapy of the diabetic foot ulcer.

Glycolytic Activation Promotes PDGF-B Production in Adipose Tissue Macrophages during Obesity Development

The vascular network develops with remodeling in adipose tissue (AT) during obesity. We have found that PDGF-B detaches pericytes from the vasculature, which plays a crucial role in angiogenesis during obesity. The expression of PDGF-B was significantly elevated in obesity-associated infiltrated CD11c-positive (+) AT macrophages (ATMs). Interestingly, the expression was low in both CD11c+ ATMs and CD206+ ATMs of lean mice. Since glycolytic and oxidative metabolism are key metabolic programs that regulate ATMs’ polarity and function, we hypothesized that hyperglycemia and inflammation synergistically drive PDGF-B production by reprogramming metabolic pathways in ATMs. The expression of PDGF-B was increased in the AT of high-fat diet (HFD)-fed mice and STZ-induced hyperglycemic mice. In accordance, exposure to high glucose led to increase the expression of PDGF-B in association with that of Itgax (CD11c) in RAW264.7 macrophages. In peritoneal macrophages, LPS but not IL-4 provoked PDGF-B expression, which was completely blocked by pretreatment with 2-DG, a glycolysis inhibitor. Moreover, LPS effectively augmented high glucose-induced its expression, and that was abolished by MG132, an NFκB inhibitor. Since macrophages highly accumulated in obese AT, where pericytes were detached from vasculature, we examined directly whether PDGF-B from macrophage involves in AT expansion during obesity. To this end, we intraperitoneally injected liposome-encapsulated clodronate (CL) to mice twice a week for 6 weeks during HFD feeding. ATMs were effectively depleted by CL with reduced PDGF-B expression in AT of HFD-induced obese mice. Importantly, HFD-induced obesity was effectively prevented by CL. In conclusion, PDGF-B from ATMs has a crucial function in AT expansion during obesity. Hyperglycemia and chronic inflammation reprogram metabolic pathways toward glycolysis, and subsequent activation of NFκB plays a pivotal role in the production of PDGF-B in ATMs during obesity. Disclosure Y. Onogi: None. T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. T. Matsuzawa: None. A. Okekawa: None. E. Watanabe: None. H. Tsuneki: None. T. Sasaoka: None.

The SDF1-CXCR4 Signals Regulate Adipose Tissue Expansion by Modulating Angiogenesis in Diet-Induced Obesity in Mice

Adipose tissue (AT) expands with angiogenesis in obesity. Mature vessels in the epididymal white adipose tissue (eWAT) are tightly wrapped with pericytes (PCs), which attenuates endothelial (ECs) proliferation. We found that obesity-associated increase of PDGF-B promoted detachment of PCs from nature vessels; thereby vessels were prone to neoangiogenesis. In this study, we aimed to clarify the role of stromal cell derived factor 1 (SDF1), another proangiogenic factor in the AT development in obesity. In the whole-mount immunofluorescence study of organ cultured eWAT, treatment with PDGF-B or SDF1 for 24 h significantly promoted detachment of PCs from vessels. The effect of SDF1 on PCs was reproduced by treatment with ATI2341, a potent CXCR4 agonist, but not by VUX11207, a CXCR7 agonist. Interestingly, PCs were well associated with vessels when tissue was treated with both PDGF-B and SDF1. In addition, the association was declined by preincubation with AMD3100, a CXCR4 antagonist or when AT was derived from Pdgfrb knockout mice. To further explore the in vivo effect of SDF1 in AT during obesity, we administrated Anagliptin (Ana), a DPP-4 inhibitor to C57BL/6J mice fed chow or HFD for 12 weeks, since SDF1 is a known substrate for DPP-4. The protein amount of SDF1 was significantly elevated in the eWAT of Ana-treated mice. Importantly, HFD-induced adipose tissue expansion was significantly attenuated with reduced vascular density and PCs detachment in the eWAT by Ana. To clarify the mechanism by which PCs attach even under HFD-fed condition by coexistence of SDF1 and PDGF-B, we isolated ECs, PCs, and macrophages (Mφ) from eWAT of mice. Expression of PDGFB was increased in Mφ and decreased in ECs by HFD, whereas these changes were attenuated and the gradient of PDGFB concentration between PCs and ECs was blunted in Ana-treated eWAT. These findings suggest that both PDGF-B and SDF1 coordinately regulate PCs detachments and adipose tissue expansion with obesity in mice. Disclosure T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. E. Watanabe: None. Y. Kotera: None. Y. Onogi: None. H. Tsuneki: None. T. Sasaoka: None.

ZNF750 inhibited the malignant progression of oral squamous cell carcinoma by regulating tumor vascular microenvironment.

Squamous cell carcinoma is often associated with the deletion or mutation of zinc finger protein 750 (ZNF750), its deletion or mutation is associated with squamous epithelial malignant biological characteristics. The present study is to explore the mechanism of ZNF750 to suppress the tumor malignant process by regulation tumor microenvironment. To evaluate the changes of tumor microenvironment in oral squamous cells carcinoma cell line CAL-27 cell, the expression of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1) and CD44 were detected by Western-blot. The changes of platelet derived growth factor (PDGFB) and tumor vascular marker CD105 (Endoglin) mRNA were estimated by qPCR. The effect of over-expressed ZNF750 on cell viability and lateral migration capacity was investigated by CCK-8 and cell scratch assay in three oral squamous cells carcinoma. ZNF750 could effectively inhibit the protein or mRNA expression of angiogenin, VEGF, RGS5 and CD105, repressed the cell adhesion molecules ITGA5, ITGB1 and CD44, but up-regulate the protein or mRNA expression of PHD2 and PDGFB. The cell viability and lateral migration ability of three oral squamous cells carcinoma were reduced by over-expression of ZNF750. ZNF750 could modulate the tumor vascular microenvironment to inhibit the oral squamous cells carcinoma malignant progression.

Inhibition Mechanism of Acellular Dermal Matrix on Capsule Formation in Expander-Implant Breast Reconstruction After Postmastectomy Radiotherapy.

Capsular contracture is one of the most common complications of expander-implant breast reconstruction. Recently, clinical reports have shown that use of an acellular dermal matrix (ADM) to cover breast implants decreases incidence of capsular contracture, but the underlying mechanism is unclear. Here, we examine how ADM reduces capsular formation in expander-implant breast reconstruction and identify cellular and molecular mechanisms of ADM-mediated reduction of capsular contracture in nonirradiated and irradiated patients. Thirty patients who underwent immediate two-stage implant-based breast reconstruction were included; 15 received radiotherapy. While the tissue expander was changed to permanent silicone implant, biopsies of the subpectoral capsule and ADM capsule were performed. Capsule thickness, immunohistochemistry of α-smooth muscle actin (αSMA), vimentin, CD31, F4/80 expression, αSMA and CD31 coexpression, and relative gene expression levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-B were investigated. Irradiated submuscular capsules were thicker than nonirradiated submuscular capsules, but the thickness of ADM capsules did not significantly differ between nonirradiated and irradiated groups. Levels of myofibroblasts, fibroblasts, vascularity, EndoMT, and macrophages were significantly lower in ADM capsules than in submuscular capsules. With the exception of EndoMT, all others were increased in irradiated submuscular capsules compared with nonirradiated submuscular capsule, while none significantly differed between nonirradiated and irradiated ADM capsules. Use of ADM reduced myofibroblasts, vascularity, fibroblasts, and EndoMT in capsule tissues. Moreover, ADM use decreased macrophages, a key regulator of tissue fibrosis, as well as TGF-β1 and PDGF-B expression. We hope that these results provide basic concepts important for prevention of capsular contracture.

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model.

Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.

Alteration of Angiogenic Gene Expression in Heart, Muscle and Adipose Tissue by Exercise

PURPOSE Exercise is one of the strongest stimuli for the induction of angiogenesis that is defined as a formation of new blood vessel. However, underlying mechanism of exercise-induced angiogenesis is not fully uncovered. Therefore, we reviewed the effect of exercise on angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin (ANG) and platelet-derived growth factor (PDGF) in heart, skeletal muscle and adipose tissue. METHODS In this review, we summarized the function of angiogenic factors and their roles in heart, skeletal muscle and adipose tissue.Also we discussed the alteration of angiogenic gene expression after exercise in three organs mentioned above. RESULTS Angiogenic regulators play a critical role in cardiac development, skeletal muscle regeneration and adipose tissue metabolism. Exercise-induced angiogenic factors contributes to the enhancement of vascular remodeling, mobilization of endothelial precursor cell and systemic metabolism. In terms of VEGF, its induction after exercise is regulated mainly by HIF-α and PGC1-α, transcriptional factors of VEGF, whereas the mechanism of exercise-induced ANG and PDGF-B expression is not clear yet. CONCLUSIONS Acute exercise, resistance exercise and endurance training increase VEGF, ANG1, ANG2 and PDGF-B expression. Thereby, exercise eventually prevents diseases including cardiovascular impairment, muscle atrophy and metabolic syndrome by increasing or maintaining blood vessel density in heart, skeletal muscle and adipose tissue. 색인어: 운동, í˜ˆê´€ì‹ ìƒ, VEGF, Angiopoietin, PDGF Keywords: Exercise, Angiogenesis, VEGF, Angiopoietin, PDGF

Co-expression of PDGF-B and VEGFR-3 strongly correlates with poor prognosis in hepatocellular carcinoma patients after hepatectomy

The ability to evaluate the prognosis of hepatocellular carcinoma (HCC) patients following hepatectomy with biological markers is of great importance. In this study, we collected samples from 90patients with HCC after hepatectomy. Immunohistochemistry was used to detect the expression of PDGF-B and VEGFR-3 in these HCC samples. According to the immunohistochemical results, PDGF-B and VEGFR-3 staining were significantly associated with clinical features. Additionally, a significant association between high PDGF-B and VEGFR-3 levels and shorter overall survival was noted, when PDGF-B and VEGFR-3 co-expression been analyzed. These results suggest that the correlative expression level of PDGF-B and VEGFR-3 has strong value in the prognosis of HCC patients following hepatectomy.

IGF-1 promotes angiogenesis in endothelial cells/adipose-derived stem cells co-culture system with activation of PI3K/Akt signal pathway.

The aim of this study was to investigate the role of insulin-like growth factor-1 (IGF-1) and crosstalk between endothelial cells (ECs) and adipose-derived stem cells (ASCs) in the process of angiogenesis. A three-dimensional collagen gel used to culture mouse ASCs and mouse ECs in vitro was established. The effects of angiogenesis after exposure to IGF-1 were observed by confocal laser scanning microscopy. Western blotting and qPCR were performed to elucidate the underlying mechanisms. IGF-1 treatment promoted the formation of vessel-like structures and the recruitment of ASCs in the three-dimensional collagen gel. The angiogenic genes and proteins in ECs were up-regulated by IGF-1 and in co-culture. Similar changes in the genes and in the proteins were detected in ASCs after exposure to IGF-1 and co-culture. p-Akt expression levels were high in ECs and ASCs after exposure to IGF-1 and co-culture. IGF-1 and co-culture between cells facilitate the process of angiogenesis via the PI3-kinase/Akt signalling pathway. In ECs, IGF-1 stimulates the expression of angiogenesis-related growth factors with the activation of the PI3-kinase/Akt signalling pathway. Co-cultured ECs exposed to excess VEGF-A and other angiogenesis-related growth factors para-secreted from ASCs exhibit high expression of angiogenesis-related genes and proteins. In ASCs, IGF-1 induces the recruitment and function of ASCs by up-regulating the expression of PDGFB, MMPs and α-SMA. Crosstalk with ECs further facilitates changes in ASCs.

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property.

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-β exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-β- MSCs. Notably, PDGFR-β+ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 106 GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-β+ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92±0.31×105 remained in wound by 7days) and accelerated wound closure; meanwhile, PDGFR-β- MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46±0.26×105) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-β+ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-β+ MSCs migrated eightfold more than PDGFR-β- MSCs toward PDGF-BB. Intriguingly, PDGFR-β+ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-β- MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-β identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.