Derived Growth Factor Receptor Beta Research Articles

Abstract 283: Fibroblast diversity and immune microenvironment in pseudoangiomatous stromal hyperplasia (PASH): Associations with breast cancer risk

Abstract Benign breast disease (BBD) affects over one million women annually in the United States and is diagnosed in nearly half of all women during their lifetime. Pseudoangiomatous stromal hyperplasia (PASH), a common BBD subtype, is characterized by an overaccumulation of tissue fibroblasts. Current research suggests a pivotal role of the immune microenvironment in progression of BBD to breast cancer, suggesting its composition as a determinant of breast cancer risk. Our study evaluates whether fibroblast variations and immune microenvironment differences in BBD biopsies containing PASH lesions are associated with subsequent breast cancer development. We evaluated BBD biopsies from women who subsequently developed breast cancer (cases, N=5) or remained cancer-free (controls, N=5). Using Opal-based multiplex immunofluorescence (mIF) assays, we mapped expression of fibroblast markers linked to breast cancer associated fibroblasts (CAFs), including vimentin (VIM), smooth muscle actin (ACTA2), fibroblast specific protein-1 (S100A4/FSP1), fibroblast associated protein (FAP), integrin beta-1 (CD29/ITGB1), and platelet derived growth factor receptor beta (PDGFRβ) and markers of key immune cells (T cells: CD4, CD8; dendritic cells: CD11c; B cells: CD20; and macrophages: CD68) along with MKI67 as a proliferation marker. Our results revealed marked differences in fibroblast marker expression and immune cell populations between these groups in the normal lobules in the BBD biopsies. PASH cases showed elevated PDGFRβ and CD29/ITGB1 expression in VIM-positive fibroblasts, markers characteristic of CAF subtypes known to accumulate in more aggressive tumor microenvironments. Conversely, a higher percentage of FSP1+/VIM+ cells, similar to that of a different CAF subtype, was noted in the lobules of PASH controls. This difference in biomarker expression, along with corresponding variations in immune cell counts, suggests a complex interplay between fibroblast composition and immune response that ultimately modulate cancer risk in PASH-containing BBD tissues. Additionally, increased MKI67 expression in PASH cases potentially signals the higher likelihood of cancer development. This work advances our understanding of fibroblast heterogeneity in BBD and its implications for breast cancer risk and progression, and paves the way for innovative strategies to improve clinical outcomes for BBD patients to reduce their incidence of breast cancer. Citation Format: Nicole Cruz-Reyes, Matilde Rossi, Melody Stallings Mann, Bryan McCauley, Tanya Hoskin, Robert Vierkant, Stacey Winham, Amy Degnim, Mark E. Sherman, Derek C. Radisky. Fibroblast diversity and immune microenvironment in pseudoangiomatous stromal hyperplasia (PASH): Associations with breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 283.

Quantitative proteomics reveals the protective effects of Yinchenzhufu decoction against cholestatic liver fibrosis in mice by inhibiting the PDGFRβ/PI3K/AKT pathway.

Introduction: Yinchenzhufu decoction (YCZFD) is a traditional Chinese medicine formula with hepatoprotective effects. In this study, the protective effects of YCZFD against cholestatic liver fibrosis (CLF) and its underlying mechanisms were evaluated. Methods: A 3, 5-diethoxycarbonyl-1, 4-dihydro-collidine (DDC)-induced cholestatic mouse model was used to investigate the amelioration of YCZFD on CLF. Data-independent acquisition-based mass spectrometry was performed to investigate proteomic changes in the livers of mice in three groups: control, model, and model treated with high-dose YCZFD. The effects of YCZFD on the expression of key proteins were confirmed in mice and cell models. Results: YCZFD significantly decreased the levels of serum biochemical, liver injury, and fibrosis indicators of cholestatic mice. The proteomics indicated that 460 differentially expressed proteins (DEPs) were identified among control, model, and model treated with high-dose YCZFD groups. Enrichment analyses of these DEPs revealed that YCZFD influenced multiple pathways, including PI3K-Akt, focal adhesion, ECM-receptor interaction, glutathione metabolism, and steroid biosynthesis pathways. The expression of platelet derived growth factor receptor beta (PDGFRβ), a receptor associated with the PI3K/AKT and focal adhesion pathways, was upregulated in the livers of cholestatic mice but downregulated by YCZFD. The effects of YCZFD on the expression of key proteins in the PDGFRβ/PI3K/AKT pathway were further confirmed in mice and transforming growth factor-β-induced hepatic stellate cells. We uncovered seven plant metabolites (chlorogenic acid, scoparone, isoliquiritigenin, glycyrrhetinic acid, formononetin, atractylenolide I, and benzoylaconitine) of YCZFD that may regulate PDGFRβ expression. Conclusion: YCZFD substantially protects against DDC-induced CLF mainly through regulating the PDGFRβ/PI3K/AKT signaling pathway.

Cross-talk between gastric cancer and hepatic stellate cells promotes invadopodia formation during liver metastasis.

In gastric cancer (GC), the liver is a common organ for distant metastasis, and patients with gastric cancer with liver metastasis (GCLM) generally have poor prognosis. The mechanism of GCLM is unclear. Invadopodia are special membrane protrusions formed by tumor cells that can degrade the basement membrane and ECM. Herein, we investigated the role of invadopodia in GCLM. We found that the levels of invadopodia-associated proteins were significantly higher in liver metastasis than in the primary tumors of patients with GCLM. Furthermore, GC cells could activate hepatic stellate cells (HSCs) within the tumor microenvironment of liver metastases through the secretion of platelet-derived growth factor subunit B (PDGFB). Activated HSCs secreted hepatocyte growth factor (HGF), which activated the MET proto-oncogene, MET receptor of GC cells, thereby promoting invadopodia formation through the PI3K/AKT pathway and subsequently enhancing the invasion and metastasis of GC cells. Therefore, cross-talk between GC cells and HSCs by PDGFB/platelet derived growth factor receptor beta (PDGFRβ) and the HGF/MET axis might represent potential therapeutic targets to treat GCLM.

Hypereosinophilic Syndrome with Endomyocarditis: Identification by Next-Generation Sequencing of the JAK2-V617F Mutation.

Hypereosinophilic syndrome requires a peripheral absolute eosinophil count of ≥1.5 × 109 /L with clinical manifestations attributable to peripheral or tissue hypereosinophilia. Clinical manifestations can vary greatly, with the majority of patients relatively asymptomatic and the eosinophilia detected incidentally. However, in a minority of hypereosinophilia cases, they may present with severe lifethreatening organ dysfunction affecting skin, lung, heart, gastrointestinal tract, and nervous system. A case of hypereosinophilia with potentially life-threatening cardiovascular involvement is discussed. Initial laboratory investigations showed an elevated white blood cell count with 60% eosinophils. An endomyocardial biopsy revealed eosinophilic endomyocarditis with granuloma, rare giant cells, and no vasculitis, microorganisms, or malignancy. Her presentation met the criteria for either hypereosinophilic syndrome or eosinophilic granulomatosis with polyangitis. Molecular genetic analysis was negative for myelodysplastic syndrome panel/ Platelet Derived Growth Factor Receptor Beta (PDGFRB) (5q32)/Fibroblast Growth Factor Receptor 1 (FGFR1) Fluorescence In Situ Hybridization (FISH), Feline McDonough Sarcoma-related Tyrosine Kinase 3 (FLT3) Internal Tandem Duplication (ITD) mutation, Calregulin (CALR) exon 9 mutation, and T-cell gene rearrangement/polymerase chain reaction. Bone marrow biopsy revealed a mildly hypocellular marrow with multilineage hematopoiesis,+ megakaryocyte dysplasia, and focal eosinophilia. No excess blasts, no monotypic B-cell population, and no discrete pan T-cell aberrancies were found. Bone marrow cytogenetic studies showed a normal signal pattern for myeloproliferative neoplasms panel/Sec1 Family Domain Containing 2 (SCFD2)-Ligand of Numb Protein-X (LNX)-Platelet-derived Growth Factor Receptor Alpha (PDGFRA) fluorescence in situ hybridization with a normal karyotype of 46 XX. Next-generation sequencing, however, was positive for the JAK2-V617F mutation, a rare molecular abnormality in hypereosinophilic syndrome. The prevalence ranges from approximately 0% to 4%. The JAK2 point mutation leads to aberrant tyrosine phosphorylation and increased cytokine activation. The case demonstrates the complexity and challenging nature of advanced diagnostic opportunities in hypereosinophilia and the potential use, in select subsets, of targeted treatments such as tyrosine kinase inhibitors.

PDGFRB upregulation contributes to retinal damages in the rat model of retinal ischemia-reperfusion

Retinal ischemic disease is a major type of retinal diseases causing vision loss. Identifying the molecular mechanisms mediating the retinal ischemia-reperfusion (RIR) is the key to targeted intervention. In this study, we performed RNA-seq analysis of the retinal tissues of a retinal ischemia-reperfusion model of Sprague-Dawley (SD) rats, followed by differential gene expression analysis, gene ontology (GO) enrichment analysis, and protein-protein interaction (PPI) analysis. After studying we found that: The major biological processes affected after RIR was the regulation of vascular development. PPI analysis unveiled a regulatory module in which Platelet Derived Growth Factor Receptor Beta (PDGFRB) was upregulated. In the RIR cell model of human retinal microvascular endothelial cells (HRCEC) induced by oxygen-glucose deprivation/reperfusion (OGD/R), silencing PDGFRB at least partially rescued the detrimental effect on cell proliferation and in vitro angiogenic ability. In the rat model of RIR, the administration of PDGFR inhibitor alleviated the damages in the retinal microvascular system. Besides, we further demonstrated the protective effect of procyanidin against RIR induced damages in both the cell and animal model by dampening the overexpression of PDGFRB. Together, our data indicate that the upregulation of PDGFRB contributes to RIR-induced damages in retinal microvascular system, which provides a targetable strategy for therapeutic intervention.

Age-dependent Pdgfrβ signaling drives adipocyte progenitor dysfunction to alter the beige adipogenic niche in male mice

Perivascular adipocyte progenitor cells (APCs) can generate cold temperature-induced thermogenic beige adipocytes within white adipose tissue (WAT), an effect that could counteract excess fat mass and metabolic pathologies. Yet, the ability to generate beige adipocytes declines with age, creating a key challenge for their therapeutic potential. Here we show that ageing beige APCs overexpress platelet derived growth factor receptor beta (Pdgfrβ) to prevent beige adipogenesis. We show that genetically deleting Pdgfrβ, in adult male mice, restores beige adipocyte generation whereas activating Pdgfrβ in juvenile mice blocks beige fat formation. Mechanistically, we find that Stat1 phosphorylation mediates Pdgfrβ beige APC signaling to suppress IL-33 induction, which dampens immunological genes such as IL-13 and IL-5. Moreover, pharmacologically targeting Pdgfrβ signaling restores beige adipocyte development by rejuvenating the immunological niche. Thus, targeting Pdgfrβ signaling could be a strategy to restore WAT immune cell function to stimulate beige fat in adult mammals.

Transcriptome Analysis of Goat Mammary Gland Tissue Reveals the Adaptive Strategies and Molecular Mechanisms of Lactation and Involution.

To understand how genes precisely regulate lactation physiological activity and the molecular genetic mechanisms underlying mammary gland involution, this study investigated the transcriptome characteristics of goat mammary gland tissues at the late gestation (LG), early lactation (EL), peak lactation (PL), late lactation (LL), dry period (DP), and involution (IN) stages. A total of 13,083 differentially expressed transcripts were identified by mutual comparison of mammary gland tissues at six developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism make adaptive transcriptional changes to meet the needs of mammary lactation. Notably, platelet derived growth factor receptor beta (PDGFRB) was screened as a hub gene of the mammary gland developmental network, which is highly expressed during the DP and IN. Overexpression of PDGFRB in vitro could slow down the G1/S phase arrest of goat mammary epithelial cell cycle and promote cell proliferation by regulating the PI3K/Akt signaling pathway. In addition, PDGFRB overexpression can also affect the expression of genes related to apoptosis, matrix metalloproteinase family, and vascular development, which is beneficial to the remodeling of mammary gland tissue during involution. These findings provide new insights into the molecular mechanisms involved in lactation and mammary gland involution.

Girdin Promotes Tumorigenesis and Chemoresistance in Lung Adenocarcinoma by Interacting with PKM2.

Girdin, an Akt substrate, has been reported to promote tumorigenesis in various tumors. However, the role of Girdin in a spontaneous tumor model has not yet been explored. Here, we studied the role of Girdin in lung adenocarcinoma (LUAD) using the autochthonous mouse model and found that Girdin led to LUAD progression and chemoresistance by enhancing the Warburg effect. Mechanistically, Girdin interacted with pyruvate kinase M2 (PKM2), which played a vital role in aerobic glycolysis. Furthermore, Girdin impaired Platelet Derived Growth Factor Receptor Beta (PDGFRβ) degradation, which in turn, promoted PKM2 tyrosine residue 105 (Y105) phosphorylation and inhibited PKM2 activity, subsequently promoting aerobic glycolysis in cancer cells. Taken together, our study demonstrates that Girdin is a crucial regulator of tumor growth and may be a potential therapeutic target for overcoming the resistance of LUAD cells to chemotherapy.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Chorioamnionitis Causes Kidney Inflammation, Podocyte Damage, and Pro-fibrotic Changes in Fetal Lambs.

BackgroundPerinatal complications, such as prematurity and intrauterine growth restriction, are associated with increased risk of chronic kidney disease. Although often associated with reduced nephron endowment, there is also evidence of increased susceptibility for sclerotic changes and podocyte alterations. Preterm birth is frequently associated with chorioamnionitis, though studies regarding the effect of chorioamnionitis on the kidney are scarce. In this study, we aim to unravel the consequences of premature birth and/or perinatal inflammation on kidney development using an ovine model.MethodsIn a preterm sheep model, chorioamnionitis was induced by intra-amniotic injection of lipopolysaccharide (LPS) at either 2, 8, or 15 days prior to delivery. Control animals received intra-amniotic injections of sterile saline. All lambs were surgically delivered at 125 days’ gestation (full term is 150 days) and immediately euthanized for necropsy. Kidneys were harvested and processed for staining with myeloperoxidase (MPO), Wilms tumor-1 (WT1) and alpha-smooth muscle actine (aSMA). mRNA expression of tumor necrosis factor alpha (TNFA), Interleukin 10 (IL10), desmin (DES), Platelet derived growth factor beta (PDGFB), Platelet derived growth factor receptor beta (PDGFRB), synaptopodin (SYNPO), and transforming growth factor beta (TGFB) was measured using quantitative PCR.ResultsAnimals with extended (but not acute) LPS exposure had an inflammatory response in the kidney. MPO staining was significantly increased after 8 and 15 days (p = 0.003 and p = 0.008, respectively). Expression of TNFA (p = 0.016) and IL10 (p = 0.026) transcripts was increased, peaking on day 8 after LPS exposure. Glomerular aSMA and expression of TGFB was increased on day 8, suggesting pro-fibrotic mesangial activation, however, this was not confirmed with PDFGB or PDGFRB. The number of WT1 positive nuclei in the glomerulus, as well as expression of synaptopodin, decreased, indicating podocyte injury.ConclusionWe report that, in an ovine model of prematurity, LPS-induced chorioamnionitis leads to inflammation of the immature kidney. In addition, this process was associated with podocyte injury and there are markers to support pro-fibrotic changes to the glomerular mesangium. These data suggest a potential important role for antenatal inflammation in the development of preterm-associated kidney disease, which is frequent.

Adolescent intermittent ethanol exposure produces Sex-Specific changes in BBB Permeability: A potential role for VEGFA

Binge drinking that typically begins during adolescence can have long-lasting neurobehavioral consequences, including alterations in the central and peripheral immune systems. Central and peripheral inflammation disrupts blood–brain barrier (BBB) integrity and exacerbates pathology in diseases commonly associated with disturbed BBB function. Thus, the goal of the present studies was to determine long-lasting effects of adolescent intermittent ethanol (AIE) on BBB integrity. For AIE, male and female Sprague Dawley rats were repeatedly exposed to ethanol (4 g/kg, intragastrically) or water during adolescence between postnatal day (P) 30 and P50. In adulthood (∼P75), rats were challenged with fluorescein isothiocyanate (FITC)-tagged Dextran of varying molecular weights (4, 20, & 70 kDa) for assessment of BBB permeability using gross tissue fluorometry (Experiment 1). Experiment 2 extended these effects using immunofluorescence, adding an adult ethanol-exposed group to test for a specific developmental vulnerability. Finally, as a first test of hypothesized mechanism, Experiment 3 examined the effect of AIE on Vascular Endothelial Growth Factor A (VEGFA) and its co-localization with pericytes (identified through expression of platelet derived growth factor receptor beta (PDGFRβ), a key regulatory cell embedded within the BBB. Male, but not female, rats with a history of AIE showed significantly increased dextran permeability in the nucleus accumbens (NAc), cingulate prefrontal cortex (cPFC), and amygdala (AMG). Similar increases in dextran were observed in the hippocampus (HPC) and ventral tegmental area (VTA) of male rats with a history of AIE or equivalent ethanol exposure during adulthood. No changes in BBB permeability were evident in females. When VEGFa expression was examined, male rats exposed to AIE were challenged with 3.5 g/kg ethanol (i.p.) or vehicle acutely in adulthood to assess long-lasting versus acute actions of ethanol. Adult rats with a history of AIE showed significantly fewer total cells expressing VEGFa in the AMG and dHPC following the acute ethanol challenge in adulthood. They also showed a significant reduction in the number of PDGFRβ positive cells that also expressed VEGFa signal. The anatomical distribution of these effects corresponded with increased BBB permeability after AIE (i.e., differential effects in the PVN, AMG, and dHPC). These studies demonstrated sex-specific effects of AIE, with males, but not females, demonstrating long-term increases in BBB permeability that correlated with changes in VEGFa and PDGFRβ protein, two factors known to influence BBB permeability.

Dasatinib causing Intracerebral Bleeding in a Patient with Chronic Myeloid Leukaemia

Dasatinib is a potent inhibitor of BCR-ABL (break point cluster-Abelson) kinase, Src family of kinases, C-kit, and Platelet Derived Growth Factor Receptor beta (PDGFR-beta), and used in the treatment of Chronic Myeloid Leukaemia (CML), Ph-positive acute lymphoblastic leukaemia, and Acute Myeloid Leukaemia (AML). The most common side effects of dasatinib are myelosuppression, gastrointestinal disturbance, fluid retention, cutaneous eruption, and bleeding diathesis. This report is about a recently diagnosed, 45-year-old female with CML, BCR-ABL positive. After six-month therapy with imatinib, the patient developed resistance to imatinib. The treatment was changed to dasatinib 70 mg, once a day. Three months after starting the therapy, the patient showed a cytological response. While receiving dasatinib, she complained of headaches, nausea, and vomiting. Her complete blood count was within the normal limit. Coagulogram was within the normal limit. Non Contrast Computerised Tomography (NCCT) of the head showed intracerebral bleed in the right frontal area of the brain. The patient was then treated with mannitol and put on artificial ventilation. She succumbed on day fourth of the Intensive Care Unit (ICU). Dasatinib has been associated with impaired platelet aggregation, and can show fatal bleeding manifestations

Multiple cell types contribute to the atherosclerotic lesion fibrous cap by PDGFRβ and bioenergetic mechanisms.

Stable atherosclerotic plaques are characterized by a thick extracellular matrix (ECM)-rich fibrous cap populated by protective ACTA2+ myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMC). Herein, we show that in murine and human lesions, 20 to 40% of ACTA2+ fibrous caps cells, respectively, are derived from non-SMC sources, including endothelial cells (EC) or macrophages that have undergone Endothelial-to-Mesenchymal (EndoMT) or Macrophage-to-Mesenchymal (MMT) transitions. In addition, we show that SMC-specific knockout of the platelet derived growth factor receptor beta (PDGFRB) in Apoe−/− mice fed a Western diet (WD) for 18 weeks resulted in brachiocephalic artery (BCA) lesions nearly devoid of SMC but with no changes in lesion size, remodeling, or indices of stability including percent of ACTA2+ fibrous cap cells. However, prolonged WD feeding of SMC-PDGFRB knockout mice resulted in reduced indices of stability, indicating that EndoMT and MMT-derived MFs cannot compensate indefinitely for loss of SMC-derived MFs. Using single cell and bulk RNA-seq analyses of the BCA region and in vitro models, we provide evidence that SMC to MF transitions (SMC-MFT) are induced by PDGF and TFGβ and dependent on aerobic glycolysis, while EndoMT is induced by IL1β and TGFβ. Together, we provide evidence that the ACTA2+ fibrous cap originates from a tapestry of cell types, which transition to an MF state through distinct signaling pathways that are either dependent on or associated with extensive metabolic reprogramming.

In‐silico high throughput whole transcriptome screening implicates cardiovascular disease and the immune system in the mechanism of action underlying adverse effects of atypical antipsychotics

AbstractBackgroundRisks of stroke/thromboembolic events, infections and death are all significantly increased by antipsychotics in people with dementia but specific mechanisms are unclear. In a novel application of a drug repurposing paradigm, we aimed to identify candidate underlying mechanisms in‐silico by leveraging publicly available transcriptomic data.MethodsWhole transcriptome signatures were first generated for three antipsychotics (amisulpride, risperidone and volinanserin) using RNA‐sequencing in SHSY‐5Y cell lines. These compounds were chosen to represent a range of mechanisms of action relevant to clinically used compounds and novel compounds in development. An unbiased high throughput screen generated correlations between each compound and a public repository of over 100,000 human disease samples. From a long list of statistically significant hits, correlations between each antipsychotic and conditions/diseases related to known side effects of antipsychotic use in dementia were identified and gene set enrichment analysis performed.ResultsStatistically significant associations were found between antipsychotic transcriptional signatures and atherosclerosis (amisulpride p=0.002; risperidone p=6.98x10‐6; volinanserin p=5.5x10‐8), venous thromboembolism (risperidone p=8.13x10‐7; volinanserin p=0.002) and influenza (amisulpride p=0.002). Pathways enriched in antipsychotic signatures were linked to the cardiovascular system, the immune system and inflammation (including brain derived neurotrophic factor, platelet derived growth factor receptor beta, tumor necrosis factor alpha signalling).ConclusionUsing a novel, high throughput approach, these findings implicate cardiovascular disease and the immune system in the mechanisms of action of atypical antipsychotics, providing a list of priority candidate mechanisms of harm relevant to dementia research. This approach could have implications for drug safety screening of psychotropic drugs in dementia.

Characterisation of a mural cell network in the murine pituitary gland

The anterior and intermediate lobes of the pituitary are composed of endocrine cells, as well as vasculature and supporting cells, such as folliculostellate cells. Folliculostellate cells form a network with several postulated roles in the pituitary, including production of paracrine signalling molecules and cytokines, coordination of endocrine cell hormone release, phagocytosis, and structural support. Folliculostellate cells in rats are characterised by expression of S100B protein, and in humans by glial fibrillary acid protein. However, there is evidence for another network of supporting cells in the anterior pituitary that has properties of mural cells, such as vascular smooth muscle cells and pericytes. The present study aims to characterise the distribution of cells that express the mural cell marker platelet derived growth factor receptor beta (PDGFRβ) in the mouse pituitary and establish whether these cells are folliculostellate. By immunohistochemical localisation, we determine that approximately 80% of PDGFRβ+ cells in the mouse pituitary have a non-perivascular location and 20% are pericytes. Investigation of gene expression in a magnetic cell sorted population of PDGFRβ+ cells shows that, despite a mostly non-perivascular location, this population is enriched for mural cell markers but not enriched for rat or human folliculostellate cell markers. This is confirmed by immunohistochemistry. The present study concludes that a mural cell network is present throughout the anterior pituitary of the mouse and that this population does not express well-characterised human or rat folliculostellate cell markers.

Abstract 7: Nerve Growth Factor Derives From Pericytes And Smooth Muscle Cells After Extremity Trauma

Purpose: Neurotrophic factors like nerve growth factor (NGF) have been described in the literature as a crucial regulator in developmental biology and inflammation of musculoskeletal tissues. While extremity trauma has been shown to elicit re-engagement of developmental programs like NGF upregulation and invasion of new sensory nerve endings in a mature organism, the source of NGF has remained largely unknown. In a validated extremity trauma mouse model, we have previously demonstrated a significant attenuation of injury site re-innervation by sensory fibers following inhibiting of afferent neural signaling and NGF binding of its receptor TrkA. Given this significant impact of NGF on sensory reinnervation of injured soft tissues and downstream bone formation, we examined the source of NGF after extremity trauma using single cell transcriptomic and reporter proteomic technologies. Methods: A 30% dorsal burn and Achilles transection was performed. The tendon site tissues were harvested from baseline (t0) and day 3, 7, and 21 (n=3-4/group) after induction. Samples were prepared for library generation on a 10x Genomics Chromium Controller, sequenced on a Illumina HiSeq 4000, and analyzed with Cell Ranger Software for pre-processing and alignment to the mm10 genome. Downstream analyses including unsupervised clustering and canonical correlation analyses were performed with Seurat. Immunofluorescent (IF) labeling of cellular markers including αSMA was performed using NGF-eGFP reporter mice at baseline, and at 1,3 and 9 weeks after injury (n=2-3/group). Results: To localize cell specific Ngf expression from injured soft tissue, 9 cell clusters were defined across all timepoints: mesodermal (Prrx1) populations including Acta2+ pericyte/vascular smooth muscle (SMC) and Pdgfra+ mesenchymal cells, two Pecam1+ endothelium, and four inflammatory cell populations (mixed, B cell, T cell, and neutrophil). Ngf was found uniquely enriched in the pericyte/SMC cluster in a composite view. These pericytes/SMCs were found in increasing number and Ngf expression across timepoints, peaking at day 21. To further characterize this joint cluster (1273 cells), the pericyte/SMC cluster was isolated and blindly re-clustered to produce new sub-clusters, distinguishing Pdgfrb (platelet derived growth factor receptor beta)highPrrx1highAbcc9+ pericytes from Acta2highPln+Myh11high SMCs; an uncharacterized cluster with 24 cells was discarded from analysis. Of these two sub-clusters, vascular SMCs demonstrated the highest Ngf expression at baseline (mean normalized Ngf expression of 0.52 [in 41% of cells] vs 0.28 [in 23% of cells]. IF of NGF-eGFP reporter mice show robust NGF co-localization with αSMA+ SMCs. Conclusions: This is the first work characterizing the pericyte and vascular SMC as a major contributor to NGF signaling at an extremity injury site. The fine resolution defining the cellular source of NGF provides insight into potential mechanisms correlating nascent nerve and vascular growth given the intersection of these pathways at the level of pericytes and myofibroblastic SMCs that will inform future candidate therapeutics to improve extremity trauma healing and re-innervation.

Perivascular osteoprogenitors are associated with transcortical channels of long bones.

Bone remodeling and regeneration are dependent on resident stem/progenitor cells with the ability to replenish mature osteoblasts and repair the skeleton. Using lineage tracing approaches, we identified a population of Dmp1+ cells that reside within cortical bone and are distinct from osteocytes. Our aims were to characterize this stromal population of transcortical perivascular cells (TPCs) in their resident niche and evaluate their osteogenic potential. To distinguish this population from osteoblasts/osteocytes, we crossed mice containing inducible DMP1CreERT2/Ai9 Tomato reporter (iDMP/T) with Col2.3GFP reporter (ColGFP), a marker of osteoblasts and osteocytes. We observed iDMP/T+;ColGFP- TPCs within cortical bone following tamoxifen injection. These cells were perivascular and located within transcortical channels. Ex vivo bone outgrowth cultures showed TPCs migrated out of the channels onto the plate and expressed stem cell markers such as Sca1, platelet derived growth factor receptor beta (PDGFRβ), and leptin receptor. In a cortical bone transplantation model, TPCs migrate from their vascular niche within cortical bone and contribute to new osteoblast formation and bone tube closure. Treatment with intermittent parathyroid hormone increased TPC number and differentiation. TPCs were unable to differentiate into adipocytes in the presence of rosiglitazone in vitro or in vivo. Altogether, we have identified and characterized a novel stromal lineage-restricted osteoprogenitor that is associated with transcortical vessels of long bones. Functionally, we have demonstrated that this population can migrate out of cortical bone channels, expand, and differentiate into osteoblasts, therefore serving as a source of progenitors contributing to new bone formation.

Abstract P6-10-24: High stromal platelet derived growth factor receptor beta predicts absence of benefit of radiotherapy after breast conserving surgery in breast ductal carcinoma in situ within the SweDCIS randomized trial

Abstract Background: With the introduction of mammography the detection of breast ductal carcinoma in situ (DCIS) has increased four- to sevenfold. Local recurrences occur only in a minority of patients. Radiotherapy (RT) is presently used without biomarker guidance. Therefore, novel biomarkers predicting benefit of radiotherapy are warranted to prevent overtreatment. Studies on RT response-predictive biomarkers in DCIS have focused on tumor cell features and often neglected the tumor-associated stroma, despite growing evidence of its active influence on treatment response including RT. The platelet derived growth factor receptors alpha and beta (PDGFRa and -b) signaling pathways are major regulators of stroma cells. Recent studies have indicated a loss of PDGFRa in the tumor-associated stroma of DCIS while PDGFRb remains high and patients with PDGFRa(low)/PDGFRb(high) expression had a significant increased risk for any tumor recurrence. A limitation of this study was though the usage of a population-based cohort, thus not allowing firm conclusions on the impact of the marker on natural course of the disease or benefit to treatment. Purpose: To explore if stromal expression of PDGFRb serves as prognostic as well as response-predictive biomarker for RT benefit with regard to ipsilateral breast tumor recurrence (IBTR) and overall survival (OS) for DCIS patients within a large and clinically well-annotated randomized trial. Method: Immunohistochemical staining of PDGFRb was performed on full tissue sections of 705 patients from the SwedDCIS cohort, a randomized RT trial after breast-conserving surgery. Stromal PDGFRb expression was evaluated by two independent raters in up to ten high power fields within the tumor associated stroma region and scored as 0/negative, 1/low average stroma intensity, 2/moderate average stroma intensity or 3/high average stroma intensity. An average PDGFRb score was calculated for each tumor. The final data was split at median defining PDGFRb low and high patient groups. Outcomes were analyzed at 10 years of follow-up. Results: High stromal PDGFRb was correlated with young age (p=0.003 Spearman’s Rank correlation). In multivariable Cox regression including RT, age, size, screen detection and tumor margin status, the PDGFRb high patient group showed a trend to worse prognosis with regard to IBTR (HR 1.40, CI95% 0.96-2.04, p=0.08) and OS (HR 1.76, CI95% 1.00-3.09, p=0.05). Benefit of radiotherapy on IBTR (in situ and invasive) was absent in the PDGFRb high patient group (HR 0.76, CI95% 0.47-1.22, p=0.25)), but clearly detected in the PDGFRb low group (HR 0.29, CI95% 0.15-0.55, p&amp;lt;0.001) in a multivariable analysis including age, size, screen detection and tumor margin status. Interaction between RT and PDGFRb was significant (p=0.020). No differences in OS after RT were observed within the PDGFR-defined patient groups. RT benefit was also restricted to the PDGFRb low patient group in separate analyses for in situ IBTR (PDGFRb low group HR 0.27, CI95% 0.12-0.60, p=0.001, versus PDGFRb high group HR 0.66, CI95% 0.34-1.29, p=0.22) or invasive IBTR (PDGFRb low group HR 0.33, CI95% 0.11-0.98, p=0.045, versus PDGFRb high group HR 0.88, CI95% 0.45-1.73, p=0.71). Significant interactions between PDGFRb group and RT were not detected in these sub-analyses (p=0.11 for in situ and p=0.15 for invasive IBTR). Conclusion: These data suggests high stromal PDGFRb as a marker of potential clinical utility for RT resistance regarding IBTR in DCIS patients. Furthermore, the study supports the hypothesis of a fibroblast-mediated modulation of RT-sensitivity in DCIS cells. Experimental model studies, investigating the biological mechanisms and potential therapeutic relevance are therefore highly motivated. Citation Format: Carina Strell, Dick Folkvaljon, Erik Holmberg, Per Karlsson, Jonas Bergh, Troy Bremer, Lars A. Akslen, Fredrik Wärnberg, Arne Östman. High stromal platelet derived growth factor receptor beta predicts absence of benefit of radiotherapy after breast conserving surgery in breast ductal carcinoma in situ within the SweDCIS randomized trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-24.

Abstract P3-08-08: Prognostic and predictive impact of stroma cells defined by platelet derived growth factor receptor beta in early breast cancer: Results from the randomized SweBGR91RT trial

Abstract Background: Although preclinical studies have indicated that stroma cells can modulate the radiosensitivity of tumor cells, non-leukocytic stroma cells have not yet been explored as potential predictive markers for radiotherapy (RT) through systematic analyses of clinical samples. Platelet-derived growth factor receptor beta (PDGFRb) is a key regulator of fibroblasts, pericytes and smooth muscle cells. A high expression of PDGFRb in tumor-associated stroma has previously been associated with worse recurrence free and breast cancer specific survival as well as reduced tamoxifen sensitivity in invasive breast cancer. Purpose: To analyze the prognostic and predictive impact of stromal PDGFRb regarding ipsilateral breast tumor recurrence (IBTR) and any recurrence in a large randomized RT trial. Methods: Stromal PDGFRb expression was assessed by immunohistochemical staining of tissue microarrays (TMAs) with 958 tumors from the SweBCG91RT trial (Swedish Breast Cancer Group 91 Radiotherapy). The trial enrolled node-negative, stage I or IIA breast cancer patients, who were randomized to postoperative RT or not after breast conserving surgery. Stained TMAs were evaluated by two independent raters for PDGFRb staining intensity (0/negative; 1/low; 2/moderate; 3/high) and positive stroma fraction (0/0%; 1/1-10%; 2/11-50; 3/51-75%; 4/76-100%). The evaluations of both raters were averaged and the product between the two scoring metrics was formed. For final analysis the data was split in tertiles, as predefined, and referred to as PDGFRb low, medium or high score group. Outcomes were analyzed at 10 years of follow-up. Results: A high PDGFRb score was correlated with ER negativity (p=0.003,), young age (p&amp;lt;0.001), large tumor size (p=0.058) and high histological grade (p=0.067) in Spearman’s Rank test. With regard to prognosis, a significantly increased risk for any recurrence was detected in univariable analysis for patients with a medium (HR 1.57, CI95% 1.11-2.23, p=0.011) or high PDGFRb score (HR 1.49, CI95% 1.06-2.10, p=0.021) compared to the PDGFRb low score group. The significance remained for the PDGFRb medium (HR 1.47, CI95% 1.03-2.11, p=0.034) but not the PDGFRb high score group (HR 1.32, CI95% 0.93-1.88, p=0.125) in a multivariable analysis including grade, age and RT. The benefit of RT regarding the risk of IBTR was significant in the PDGFRb low (HR 0.28, CI95% 0.12-0.65, p=0.003) and medium (HR 0.32, CI95% 0.16-0.61, p=0.001) score groups but not in the PDGFRb high (HR 0.61, CI95% 0.35-1.07, p=0.086) score group in a multivariable analysis including grade and age. The risk reduction from RT regarding any recurrence was also less pronounced in the PDGFRb high (HR 0.73, CI95% 0.48-1.12, p=0.149) score group compared to the PDGFRb low (HR 0.57, CI95 0.32-1.03, p=0.065) and medium (HR 0.46, CI95% 0.28-0.75, p=0.002) score groups in multivariable analysis. No significant interaction between RT and PDGFRb expression could however be detected for IBTR (p=0.14) or any recurrence (p=0.33). Conclusion: A higher PDGFRb score conferred a negative prognosis with increased risk of any recurrence in univariable analyses, which in part can be explained by its association with ER negativity, young age and grade. A predictive effect of RT benefit from PDGFRb could not be confirmed although a tendency toward less benefit was observed in the patient group with the highest PDGFRb score. Citation Format: Carina Strell, Reidunn J Edelmann, Axel Stenmark Tullberg, Per Malmström, Mårten Fernö, Arne Östman, Erik Holmberg, Per Karlsson. Prognostic and predictive impact of stroma cells defined by platelet derived growth factor receptor beta in early breast cancer: Results from the randomized SweBGR91RT trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-08.

Non-Invasively Distinguishing Progress of Liver Fibrosis by Visualizing Hepatic Platelet Derived Growth Factor Receptor-Beta Expression with an MRI Modality in Mice

Background: Activated hepatic stellate cells (HSCs) are the most critical cell responsible for liver fibrosis. In liver fibrogenesis, platelet-derived growth factor (PDGF) is the most prominent mitogen for HSCs. This study aims to explore the potential of gadolinium (Gd)-labeled cyclic peptides (pPB) targeted to PDGF receptor-β (PDGFR-β) as an MRI radiotracer to identify the progress of liver fibrosis by imaging hepatic PDGFR-β expression. Methods: Mouse models of liver fibrosis caused by bile duct ligation (BDL) and CCl 4 treatment were both used to determine the association of hepatic PDGFR-β expression and the progress of hepatic fibrosis. The binding activity of FITC-labeled pPB to PDGFR-β was assessed in culture-activated human HSCs (HSC-LX2). MR imaging was performed to visualize hepatic PDGFR-β expression in mice with different degrees of liver fibrosis after Gd-labeled pPB were injected. Findings: Hepatic PDGFR-β expression level was found to be paralleled with the severity of liver fibrosis, which was increased with the progression of fibrosis and reduced with the regression. Majority of cells expressing PDGFR-β was determined to be activated HSCs in fibrotic livers. Culture-activated HSC-LX2 expressed abundant PDGFR-β, and FITC-labeled pPB could bind to HSC-LX2 in a concentration and time dependent manner. Interpretation: With Gd-labeled pPB as a tracer, the relative hepatic T1-weighed MR signal value was increased progressively along with severity of hepatic fibrosis and reduced with the remission, suggesting that hepatic PDGFR-β expression reflects the progress of hepatic fibrosis. MR imaging using Gd-labeled pPB as a tracer may distinguish different stages of liver fibrosis in mice. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant NO. 81270007 & 81670513 to Li F), Shanghai Talent Development Funds (Grant NO. 201304 to Li F), Shanghai Rising-Star Program (Grant NO. 13QA1400700 to Li F), And Shanghai Outstanding Young Talent Training Plan in Health System (Grant NO. 13YO56 to Li F). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: The study was approved by Institutional Ethical Committee of Animal Experimentation, all experiments and postoperative animal care procedures were performed according to the governmental and international guidelines on animal experimentation.