Platelet-derived Growth Factor Beta Research Articles

Abstract 1437: Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing

Abstract Diffuse midline gliomas (DMGs) are deadly brain tumors that arise in the brainstem of children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions in DMG, which may inform the design of new urgently-needed treatment approaches. One approach to generate DMG GEMMs uses RCAS/tv-a, an avian retrovirus gene delivery system to express transgenes in specific brain cell lineages in the mouse brain. However, this approach often requires resource-intensive breeding to generate mice with the desired genetic alterations in their germline. As a result, we aimed to establish a new approach that combines this system with CRISPR/Cas9 gene editing technology. This methodology has been described for model adult supratentorial gliomas in mice but has not been described for pediatric or brainstem gliomas. To express Cas9 and the avian retrovirus receptor TVA in mouse neural stem cells, we bred mice of genotype Nestin-TVA; Nestin-Cre; LoxP-STOP-LoxP-Cas9-EGFP. RCAS retrovirus plasmids were generated containing various guide RNAs (gRNAs) targeting DMG-relevant genes for disruption by Cas9, including Trp53, Cdkn2a, Atm, Ppm1d, Pten, and a non-targeting control (Cntl). For each gRNA, two plasmids were generated, one with a platelet-derived growth factor beta ligand (PDGFB) oncogene driver and one with a blue fluorescent protein (BFP) marker. In a pilot experiment, avian fibroblast cells transduced with RCAS-Trp53_gRNA_BFP and RCAS-Cntl_gRNA-PDGFB were injected into postnatal day 4 mouse brainstems of the aforementioned genotype and observed for tumor formation. High grade tumor formation was observed. We also observed high grade tumors in mice of genotype Nestin-TVA; LoxP-STOP-LoxP-Cas9-EGFP when injected with an additional RCAS-Cre construct, indicating that Cre can be expressed from the mouse germline or delivered via a retrovirus payload to drive tumorigenesis. Immunohistochemistry confirmed tumor features that model human DMG, expression of Cas9 and PDGFB, and loss of p53. Thus, RCAS/tv-a and CRISPR/Cas9 can be combined to model pediatric glioma tumorigenesis in the mouse brainstem. This approach to mouse modeling will streamline future GEMM DMG research by allowing new genes of interest to be rapidly perturbed by simply generating RCAS retrovirus constructs. The workflow may also enable sophisticated experiments such as pooled in vivo genetic experiments that screen gRNAs targeting various tumor suppressor genes or therapeutic targets. Citation Format: Sophie R. Wu, Harrison Q. Liu, Joshua Tolliver, Spencer M. Maingi, Vennesa Valentine, Nerissa T. Williams, Zachary J. Reitman. Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing [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 1437.

Small vessel disease in primary familial brain calcification with novel truncating PDGFB variants.

Primary familial brain calcification (PFBC) is a neurodegenerative disease characterised by bilateral calcification in the brain, especially in the basal ganglia, leading to neurological and neuropsychiatric manifestations. White matter hyperintensities (WMH) have been described in patients with PFBC and pathogenic variants in the gene for platelet-derived growth factor beta polypeptide (PDGFB), suggesting a manifest cerebrovascular process. We present below the cases of two PFBC families with PDGFB variants and stroke or transient ischaemic attack (TIA) episodes. We examine the possible correlation between PFBC and vascular events as stroke/TIA, and evaluate whether signs for vascular disease in this condition are systemic or limited to the cerebral vessels. Two Swedish families with novel truncating PDGFB variants, p.Gln140* and p.Arg191*, are described clinically and radiologically. Subcutaneous capillary vessels in affected and unaffected family members were examined by light and electron microscopy. All mutation carriers showed WMH and bilateral brain calcifications. The clinical presentations differed, with movement disorder symptoms dominating in family A, and psychiatric symptoms in family B. However, affected members of both families had stroke, TIA, and/or asymptomatic intracerebral ischaemic lesions. Only one of the patients had classical vascular risk factors. Skin microvasculature was normal. Patients with these PDGFB variants develop microvascular changes in the brain, but not the skin. PDGFB-related small vessel disease can manifest radiologically as cerebral haemorrhage or ischaemia, and may explain TIA or stroke in patients without other vascular risk factors.

Establishment and characterization of NCC-DFSP5-C1: a novel patient-derived dermatofibrosarcoma protuberans cell line.

Dermatofibrosarcoma protuberans (DFSP) is the most prevalent dermal sarcoma, characterized by the presence of the fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta chain (PDGFB) gene. Although PDGF receptor inhibitor imatinib mesylate was approved for the treating patients with unresectable or metastatic DFSP, disease progression was shown in 9.2% of the patients. Therefore, developing novel therapeutic strategies is crucial for improving the prognosis of DFSP. Patient-derived cell lines play a vital role in preclinical studies; however, only a limited number of DFSP cell lines are currently available in public cell banks. Here, we successfully established a novel DFSP cell line (NCC-DFSP5-C1) using surgically resected tumor tissue from a patient with DFSP. NCC-DFSP5-C1 cells were confirmed to carry the COL1A1-PDGFB translocation and maintain the same mutation as the original tumor tissue. They exhibited consistent growth, formed spheroids, and were invasive. By screening a drug library using NCC-DFSP5-C1 and four previously established DFSP cell lines, we identified anti-cancer drugs that inhibit DFSP cell proliferation. Our observations suggest that the NCC-DFSP5-C1 cell line holds promise as a valuable tool for conducting fundamental and preclinical studies for DFSP.

Sry-Box Transcription Factor 9 (SOX9)-Driven Mesenchymal Stromal Cell Differentiation Signature As a Determinant of Induction Chemotherapy Responsiveness in Acute Myeloid Leukemia Core Bone Marrow Biopsies

Leukemic clones in acute myeloid leukemia (AML) alter the bone marrow (BM) microenvironment so that the normal functions of hematopoietic stem cells (HSCs) are interrupted, aiding malignant progression. Accumulating evidence points to a protective role that the microenvironment provides for the AML clone (Colmone et al., Science, 2008). It is therefore crucial to detail the mechanisms by which the BM stroma promotes leukemia progression to inform the development of more effective targeted therapies. Most studies focused on detailing the role of BM stroma in AML etiology use BM aspirates, which have a limited ability to represent the physical structure of the BM microenvironment. To accurately capture BM composition, our group used the core bone marrow biopsies (cBMBs) of 15 AML patients who responded to and 14 AML patients who did not respond to 7+3 induction chemotherapy (Treaba et al., British Journal of Haematology, 2022). RNA-sequencing data of these biopsies (GEO GSE213210) indicated significant differences in the composition of the stromal component of responders and non-responders. Responder cBMBs were enriched in undifferentiated CXCL12-positive mesenchymal stem cells (MSCs), with elevated expression of platelet derived growth factor beta (PDGFRB), snail family transcriptional repressor 2 (SNAI2), or vimentin (VIM). CXCL12-positive MSCs in non-responder cBMBs had increased expression of osteo-committed markers such as osteopontin (SPP1), osterix (SP7), and collagens (Figure 1B, osteogenesis). Based on these findings, we formulated the hypothesis that the differentiation state of the BM stroma at diagnosis may indicate treatment outcomes, specifically at the initial 14-day post-induction therapy assessment. First, we determined the adipocyte- and chondrocyte-commitment states of BM MSCs in responder and non-responder AML cBMBs before and after the 14-day induction therapy. We found significant increases in transcripts linked to adipogenesis in responder cBMBs, suggesting an increase in frequencies of adipo-committed MSCs that represent the early differentiation state of these cells. This observation was consistent with our already reported findings (Treaba et al., British Journal of Haematology, 2022) that less differentiated MSCs are prevalent in responder cBMBs post-treatment. Intriguingly, we also noted significant differences between expression of SOX9-regulated transcripts in responder compared to non-responder cBMBs. As a master regulator of chondrogenesis, SOX9 is required for MSCs to differentiate towards a chondrogenic fate. SOX9 expression was significantly upregulated in non-responder post-treatment versus diagnostic cBMBs, with a log2 fold change of 3.6. Collagen 2 alpha 1 (COL2a1) and collagen 9 alpha 1 (COL9a1), two targets of SOX9 important in chondrogenesis, were also notably elevated in non-responder cBMBs with a log2 fold change of 6.1 and 7, respectively. In contrast, SOX6, which also regulates chondrogenesis, and COL2a1 were downregulated in responder post-treatment versus diagnostic cBMBs, with a log2 fold change of -2.7 and -4.5, respectively. Another striking change was seen when directly comparing the transcriptomes of responder to non-responder cBMBs at both diagnosis and post-treatment stages. Non-responder log2 expression of COL2a1 relative to responder log2 expression increased from -4.7 at diagnosis to 5.9 at post-treatment. Importantly, SOX9-regulated or -interacting genes were the most downregulated chondrogenic genes in responder cBMBs and the most upregulated in non-responder cBMBs, which is consistent with the contrasting differentiated state of MSCs between responders and non-responders initially observed with osteogenic genes. In summary, for the first time, we report that expression levels of SOX9 and SOX9-regulated targets in AML patients are indicative of the differentiated state of MSCs in AML patients who responded to or did not respond to 7+3 induction therapy. These data represent compelling support for our hypothesis correlating mesenchymal differentiation capacity with treatment response and warrant in-depth mechanistic studies.

Urine Proteomics Link Complement Activation with Interstitial Fibrosis/Tubular Atrophy in Lupus Nephritis Patients

BackgroundIntrarenal complement activation has been implicated in the pathogenesis of tubulointerstitial fibrosis in lupus nephritis (LN) based on prior animal studies. The assembly of the membrane attack complex (MAC) by complement C5b to C9 on the cell membrane leads to cytotoxic pores and cell lysis, while CD59 inhibits MAC formation by preventing C9 from joining the complex. We hypothesize that complement activation and imbalance between complement activation and inhibition, as defined by increased production of individual complement components and uncontrolled MAC activation relative to CD59 inhibition, are associated with interstitial fibrosis and tubular atrophy (IFTA) in LN and correlate with the key mediators of kidney fibrosis- transforming growth factor receptors beta (TGFRβ), platelet-derived growth factor beta (PDGFβ) and platelet-derived growth factor receptor beta (PDGFRβ). MethodsWe included urine samples from 46 adults and pediatric biopsy-proven lupus nephritis patients who underwent clinically indicated kidney biopsies between 2010 and 2019. We compared individual urinary complement components and the urinary C9-to-CD59 ratio between LN patients with moderate/severe IFTA and none/mild IFTA. IFTA was defined as none/mild (<25% of interstitium affected) versus moderate/severe (≥ 25% of interstitium affected). Proteomics analysis was performed using mass spectrometry (Orbitrap Fusion Lumos, Thermo Scientific) and processed by the Proteome Discoverer. Urinary complement proteins enriched in LN patients with moderate/severe IFTA were correlated with serum creatinine, TGFβR1, TGFβR2, PDGFβ, and PDGFRβ. ResultsOf the 46 LN patients included in the study, 41 (89.1%) were women, 20 (43.5%) self-identified as Hispanic or Latino, and 26 (56.5%) self-identified as Black or African American. Ten of the 46 (21.7%) LN patients had moderate/severe IFTA on kidney biopsy. LN patients with moderate/severe IFTA had an increased urinary C9-to-CD59 ratio [median 0.91 (0.83-1.05) vs 0.81 (0.76-0.91), p=0.01]. Urinary C3 and CFI levels in LN patients with moderate/severe IFTA were higher compared to those with none/mild IFTA [C3 median (IQR) 24.4(23.5-25.5) vs. 20.2 (18.5–22.2), p= 0.02], [CFI medium (IQR) 28.8 (21.8-30.6) vs. 20.4 (18.5-22.9), p=0.01]. Complement C9, CD59, C3 and CFI correlated with TGFβR1, PDGFβ, and PDGFRβ, while C9, CD59 and C3 correlated with TGFβR2. ConclusionThis study is one of the first to compare the urinary complement profile in LN patients with moderate/severe IFTA and none/mild IFTA in human tissues. This study identified C3, CFI, and C9-to-CD59 ratio as potential markers of tubulointerstitial fibrosis in LN.

Reversal of fibrosis and portal hypertension by Empagliflozin treatment of CCl4-induced liver fibrosis: Emphasis on gal-1/NRP-1/TGF-β and gal-1/NRP-1/VEGFR2 pathways

To date, liver fibrosis has no clinically approved treatment. Empagliflozin (EMPA), a highly selective sodium-glucose-cotransporter-2 (SGLT2) inhibitor, has shown ameliorative potential in liver diseases without revealing its full mechanisms. Neuropilin-1 (NRP-1) is a novel regulator of profibrogenic signaling pathways related to hepatic stellate cells (HSCs) and hepatic sinusoidal endothelial cells (HSECs) that modulates intrahepatic profibrogenic and angiogenic pathways. Herein, EMPA's antifibrotic potentials and effects on galactin-1 (Gal-1)/NRP-1 signaling pathways have been evaluated in an experimental liver fibrosis rat model by testing different EMPA dose regimens. EMPA treatment brought a dose-dependent decrease in Gal-1/NRP-1 hepatic expression. This was coupled with suppression of major HSCs pro-fibrotic pathways; transforming growth factor-β (TGF-β)/TGF-βRI/Smad2 and platelet-derived growth factor-beta (PDGF-β) with a diminution of hepatic Col 1A1 level. In addition, EMPA prompted a protuberant suppression of the angiogenic pathway; vascular endothelial growth factor (VEGF)/VEGF-receptor-2 (VEGFR-2)/SH2-Domain Containing Adaptor Protein-B (Shb), and reversal of altered portal hypertension (PHT) markers; endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS). The amelioration of liver fibrosis was coupled with a remarkable improvement in liver aminotransferases and histologic hepatic fibrosis Ishak scores. The highest EMPA dose showed a good safety profile with minimal changes in renal function and glycemic control. Thus, the current study brought about novel findings for a potential liver fibrosis treatment modality via targeting NRP-1 signaling pathways by EMPA.

The effect of oral treatment of royal jelly on the expression of the PDGF-β gene in the skin wound of male mice

Aims of the studyRoyal jelly (RJ) is one of the most widely used drugs in traditional medicine. One of its important applications is the repair of skin damage, although the path of its mechanism is still unknown. Platelet-derived growth factor-beta (PDGF-beta) is one of the important factors in wound healing and it has been observed that PDGF-β expression decreases with increasing age. In this study, for the first time, the effect of RJ on skin wounds has been investigated through the expression of PDGF-β and tissue studies. Materials and methods25 small laboratory male BALB/c mice were selected randomly and after creating a 5 mm wound on the back of their neck, they were treated with doses of 2.5, 10, and 40 mg/kg body weight, After sampling from the healed wound in 9th day, histopathological studies and the expression of PDGF-β gene were performed by Real-time PCR method. ResultsThe findings of the present study showed that royal jelly caused a significant increase in PDGF-β (10.99 times) compared to the healthy group. Also, royal jelly increased the formation of covering tissue or epithelium, the synthesis of collagen, the presence of inflammatory cells, and the formation of new blood vessels. ConclusionThe oral treatment of royal jelly is probably effective in skin wound healing by changing the expression of PDGF-β.

Chronic Morphine Modulates PDGFR-β and PDGF-B Expression and Distribution in Dorsal Root Ganglia and Spinal Cord in Male Rats

The analgesic effect of opioids decreases over time due to the development of analgesic tolerance. We have shown that inhibition of the platelet-derived growth factor beta (PDGFR-β) signaling eliminates morphine analgesic tolerance in rats. Although the PDGFR-β and its ligand, the platelet-derived growth factor type B (PDGF-B), are expressed in the substantia gelatinosa of the spinal cord (SG) and in the dorsal root ganglia (DRG), their precise distribution within different cell types of these structures is unknown. Additionally, the impact of a tolerance-mediating chronic morphine treatment, on the expression and distribution of PDGF-B and PDGFR-β has not yet been studied. Using immunohistochemistry (IHC), we found that in the spinal cord, PDGFR-β and PDGF-B were expressed in neurons and oligodendrocytes and co-localized with the mu-opioid receptor (MOPr) in opioid naïve rats. PDGF-B was also found in microglia and astrocytes. Both PDGFR-β and PDGF-B were detected in DRG neurons but not in spinal primary afferent terminals. Chronic morphine exposure did not change the cellular distribution of PDGFR-β or PDGF-B. However, PDGFR-β expression was downregulated in the SG and upregulated in the DRG. Consistent with our previous finding that morphine caused tolerance by inducing PDGF-B release, PDGF-B was upregulated in the spinal cord. We also found that chronic morphine exposure caused a spinal proliferation of oligodendrocytes. The changes in PDGFR-β and PDGF-B expression induced by chronic morphine treatment suggest potential mechanistic substrates underlying opioid tolerance.

Infiltration of meningeal macrophages into the Virchow-Robin space after ischemic stroke in rats: Correlation with activated PDGFR-β-positive adventitial fibroblasts.

Macrophages play a crucial role in wound healing and fibrosis progression after brain injury. However, a detailed analysis of their initial infiltration and interaction with fibroblasts is yet to be conducted. This study aimed to investigate the possible route for migration of meningeal macrophages into the ischemic brain and whether these macrophages closely interact with neighboring platelet-derived growth factor beta receptor (PDGFR-β)-positive adventitial fibroblasts during this process. A rat model of ischemic stroke induced by middle cerebral artery occlusion (MCAO) was developed. In sham-operated rats, CD206-positive meningeal macrophages were confined to the leptomeninges and the perivascular spaces, and they were not found in the cortical parenchyma. In MCAO rats, the number of CD206-positive meningeal macrophages increased both at the leptomeninges and along the vessels penetrating the cortex 1 day after reperfusion and increased progressively in the extravascular area of the cortical parenchyma by 3 days. Immunoelectron microscopy and correlative light and electron microscopy showed that in the ischemic brain, macrophages were frequently located in the Virchow-Robin space around the penetrating arterioles and ascending venules at the pial surface. This was identified by cells expressing PDGFR-β, a novel biomarker of leptomeningeal cells. Macrophages within penetrating vessels were localized in the perivascular space between smooth muscle cells and PDGFR-β-positive adventitial fibroblasts. In addition, these PDGFR-β-positive fibroblasts showed morphological and molecular characteristics similar to those of leptomeningeal cells: they had large euchromatic nuclei with prominent nucleoli and well-developed rough endoplasmic reticulum; expressed nestin, vimentin, and type I collagen; and were frequently surrounded by collagen fibrils, indicating active collagen synthesis. In conclusion, the perivascular Virchow-Robin space surrounding the penetrating vessels could be an entry route of meningeal macrophages from the subarachnoid space into the ischemic cortical parenchyma, implying that activated PDGFR-β-positive adventitial fibroblasts could be involved in this process.

Lin28b delays vasculature aging by reducing platelet-derived growth factor-beta resistance in senescent vascular smooth muscle cells

Background and aimsPlatelet-derived growth factor-β (PDGFB) is an important mediator of vascular smooth muscle cell (VSMC) proliferation, and PDGFB resistance is observed in senescent VSMCs. Lin28b is a stemness regulator in the embryo; however, its role in vasculature aging and VSMC senescence is unknown. We aimed to investigate whether Lin28b could restore the VSMC response to PDGFB and delay vasculature aging. MethodsApoE−/− mice were fed a high-fat diet for different weeks to establish an aging model. PDGFB resistance was observed using EdU staining in vessel culture in vitro. Quantitative polymerase chain reaction and in situ hybridization were used to detect let-7 expression. Senescence was identified by Western blotting, senescence-associated beta-galactosidase activity or Sudan Black B staining, and VSMC function was determined using CCK-8, migration, and enzyme-linked immunosorbent assays. ResultsVessels from aged mice showed poor responses to PDGFB stimulation compared with those from young mice; similar results were found in senescent VSMCs. The expression levels of Lin28b and PDGF receptor-β were downregulated in aging vasculature and senescent VSMCs, whereas let-7 family levels increased with aging and VSMC passage growth. Transfection of VSMCs with let-7c induced PDGFB resistance and accelerated VSMC senescence, whereas blocking let-7c restored PDGFB reactions in VSMCs. Overexpression of Lin28b protein by lentivirus resulted in the restoration of PDGFB reactions and delayed VSMC senescence, which was blocked by a let-7c mimic. ConclusionsThis study reveals the role of Lin28b in delaying vasculature aging by decreasing senescent VSMC PDGFB resistance mediated by let-7.

MODL-28. DEVELOPING A STRATEGY FOR MODELING HIGH-GRADE GLIOMA IN GӦTTINGEN MINIPIGS

Abstract BACKGROUND The current literature does not describe a reproducible large animal model of intracranial high-grade glioma (HGG). Prior work has demonstrated the feasibility of inducing HGG de-novo in rodents by targeting specific oncogenic pathways. Here we report our approach to the production of supratentorial HGG in a series of minipigs through lentiviral gene transfer and subsequent initial characterization of a porcine glioma cell line. METHODS Four minipigs received injections into the subcortical white matter using a combination of lentiviral vectors expressing platelet-derived growth factor beta (PDGF-B), HRAS, and shRNA-p53. Animals underwent behavioral monitoring through porcine neurobehavioral scoring (PNS) and veterinary monitoring. Magnetic resonance imaging (MRI) was conducted at endpoint prior to necropsy. Post-mortem tissue biopsies underwent tissue culture and neuropathologic evaluation with hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and immunofluorescent staining. Data are presented using appropriate statistical tests where relevant and descriptive statistics. RESULTS Two pigs received 50ul injections and reached endpoint by the end of post-operative week 1 and 2. Two pigs received 25 ul injections and were asymptomatic until a pre-determined endpoint of 4 weeks. MRI scans at endpoint demonstrated contrast enhancing, mass forming lesions at the site of injection with evidence of hemorrhage and perilesional edema, consistent with high-grade glioma. On H&amp;E staining high-grade glioma growth was identified in 100% of animals. We observed immunopositivity for tumor markers GFAP, OLIG2, NG2, SOX2, and PDGFRA, as well as redox markers, and microenvironmental features consistent with high-grade glioma. Porcine glioma cell cultures were found to have significantly greater proliferative rate compared to control, and demonstrated GFAP, OLIG2, PDGFRA, and CD68 immunopositivity. CONCLUSIONS Lentiviral gene transfer represents a feasible strategy for glioma modeling in the Gӧttingen minipig. With our described methodology, we present a realistic strategy for reproducible modeling of intracranial glioma as a platform for preclinical neurosurgical development programs.

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.

Platelet-derived growth factor BB is reduced in endometrial endothelial cells of women with abnormal uterine bleeding–endometrial disorder

Research questionWhat is the expression pattern of platelet-derived growth factor BB (PDGF-BB), and its receptors, across the menstrual cycle in healthy control women and those with abnormal uterine bleeding–endometrial disorder (AUB-E)? DesignImmunohistochemical staining for PDGF-BB, platelet-derived growth factor receptor alpha (PDGFRα) and platelet-derived growth factor beta (PDGFRβ) was performed in control and AUB-E endometrium from the proliferative, early, mid- and late secretory phases of the menstrual cycle (n = 5 each group). Control proliferative phase endometrium was cultured in PDGF-BB (0, 10 ng/ml) and vascular maturation assessed (n = 3). Endothelial cell to vascular smooth muscle cell (VSMC) association was assessed after treatment with PDGF-BB (0, 1, 10 ng/ml). Secretion of angiogenic growth factors by endothelial cells or VSMC was determined. ResultsEndothelial cell immunoreactivity for PDGF-BB was reduced in the mid and late secretory phases in AUB-E (P = 0.008). PDGFRα was also reduced in mid secretory phase endothelial cells, proliferative and early secretory phase glandular epithelium in AUB-E (P = 0.008). PDGFRβ expression was not altered. Treatment of proliferative phase endometrium with PDGF-BB (10 ng/ml) reduced the percentage of vessels expressing contractile VSMC markers. PDGF-BB had no effect on angiogenic growth factor secretion by endothelial cells or VSMC in vitro and did not affect their association in an in-vitro endothelial cell–VSMC association assay. ConclusionsReduced endothelial cell expression of PDGF-BB in the AUB-E endometrium may contribute to the reduced vascular maturation previously observed in these women.

Advanced platelet-rich fibrin promotes the paracrine function and proliferation of adipose-derived stem cells and contributes to micro-autologous fat transplantation by modulating HIF-1α and VEGF

BackgroundThe micro-autologous fat transplantation (MAFT) technique has demonstrated its feasibility in multiple medical fields, such as facial rejuvenation. Advanced platelet-rich fibrin (APRF), an autologous platelet concentrated on a fibrin membrane without added external factors, has shown significant potential for tissue restoration. However, the role of APRF in the modulation of MAFT remains unclear. Here, we aimed to explore the effect of APRF on MAFT.MethodsAdipose-derived stem cells (ASCs) were isolated from human gastric subcutaneous fat and treated with APRF. ELISA assays measured cytokines. The proliferation of ASCs was analyzed by CCK-8 assays. The levels of hypoxia-inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), insulin like growth factor 2 (IGF-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) were measured by ELISA assays, quantitative reverse transcription-PCR (qRT-PCR), and Western blot analysis. The effect of APRF/HIF-1α/VEGF on MAFT in vivo was analyzed in Balb/c nude mice. The BALB/c mice were subcutaneously co-transplanted with fat, APRF, and control shRNA, HIF-1α shRNA, or VEGF shRNA into the dorsal area. The serum and protein levels of the above cytokines were analyzed by ELISA assays and Western blot analysis. Lipid accumulation was measured by Oil Red O staining. The expression of CD34 was assessed by immunohistochemical staining.ResultsAPRF continuously secreted multiple cytokines, including epidermal growth factor (EGF), FGF-2, insulin like growth factor 1 (IGF-1), interleukin-1beta (IL-1β), interleukin-4 (IL-4), platelet-derived growth factor alpha polypeptide b (PDGF-AB), platelet-derived growth factor beta polypeptide b (PDGF-BB), transforming growth factor-beta (TGF-β), and VEGF. APRF was able to promote the proliferation of ASCs. APRF dose-dependently activated the expression of HIF-1α, HSP70, IGF-2, IL-6, IL-8, and VEGF in ASCs. APRF regulated the paracrine function of ASCs by modulating HIF-1α and VEGF. APRF increased the survival of MAFT by modulating HIF-1α and VEGF in vivo.ConclusionsAPRF promotes the paracrine function and proliferation of ASCs and contributes to MAFT by modulating HIF-1α and VEGF. Our findings provide new insights into the mechanism by which APRF regulates MAFT.

Integrated molecular analysis identifies a conserved pericyte gene signature in zebrafish

ABSTRACT Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, and kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.

Zebrafish Vascular Mural Cell Biology: Recent Advances, Development, and Functions.

Recruitment of mural cells to the vascular wall is essential for forming the vasculature as well as maintaining proper vascular functions. In recent years, zebrafish genetic tools for mural cell biology have improved substantially. Fluorescently labeled zebrafish mural cell reporter lines enable us to study, with higher spatiotemporal resolution than ever, the processes of mural cell development from their progenitors. Furthermore, recent phenotypic analysis of platelet-derived growth factor beta mutant zebrafish revealed well-conserved organotypic mural cell development and functions in vertebrates with the unique features of zebrafish. However, comprehensive reviews of zebrafish mural cells are lacking. Therefore, herein, we highlight recent advances in zebrafish mural cell tools. We also summarize the fundamental features of zebrafish mural cell development, especially at early stages, and functions.

Why PRP works only on certain patients with tennis elbow? Is PDGFB gene a key for PRP therapy effectiveness? A prospective cohort study

BackgroundThere is variability in individual response to platelet-rich plasma (PRP) therapy in tennis elbow treatment. Genetic variation, especially within genes encoding growth factors may influence the observed inter-individual differences. The purpose of this study was to identify polymorphic variants of the platelet-derived growth factor beta polypeptide gene (PDGFB) that determine an improved individual response to PRP therapy in tennis elbow patients.MethodsThis prospective cohort study was designed in accordance with STROBE and MIBO guidelines. A cohort of 107 patients (132 elbows, 25 bilateral) was studied, including 65 females (77 elbows) and 42 males (55 elbows), aged 24–64 years (median 46.00 ± 5.50), with lateral elbow tendinopathy treated with autologous PRP injection. The effectiveness of PRP therapy was recorded in all subjects at 2, 4, 8, 12, 24 and 52 weeks after PRP injection using the Visual Analog Scale (VAS), quick version of Disabilities of the Arm, Shoulder and Hand score (QDASH) and Patient-Rated Tennis Elbow Evaluation (PRTEE). In order to determine the PDGFB variants with the best response to PRP therapy, patient reported outcome measures were compared between individual genotypes within studied polymorphic variants (rs2285099, rs2285097, rs2247128, rs5757572, rs1800817 and rs7289325). The influence of single nucleotide polymorphisms on blood and PRP parameters, including the concentration of PDGF-AB and PDGF-BB proteins was also analyzed.ResultsOur analysis identified genetic variants of the PDGFB gene that lead to a better response to PRP therapy. The TT (rs2285099) and CC (rs2285097) homozygotes had higher concentration of platelets in whole blood than carriers of other genotypes (p = 0.018) and showed significantly (p < 0.05) lower values of VAS (weeks 2–12), QDASH and PRTEE (weeks 2–24). The rs2285099 and rs2285097 variants formed strong haplotype block (r2 = 98, D’=100). The AA homozygotes (rs2247128) had significantly lower values of VAS (weeks 4–52), QDASH and PRTEE (weeks 8, 12).ConclusionsPDGFB gene’s polymorphisms increase the effectiveness of PRP therapy in tennis elbow treatment. Genotyping two polymorphisms of the PDGFB gene, namely rs2285099 (or rs2285097) and rs2247128 may be a helpful diagnostic tool while assessing patients for PRP therapy and modifying the therapy to improve its effectiveness.

Determination of COL1A1–PDGFB breakpoints by next-generation sequencing in the molecular diagnosis of dermatofibrosarcoma protuberans

ObjectiveIn most cases, dermatofibrosarcoma protuberans (DFSP) is characterized by the chromosomal translocation t (17; 22) (q22; q13) that leads to a fusion of collagen type 1 alpha 1 (COL1A1) and platelet-derived growth factor beta chain (PDGFB). Recently, next-generation sequencing (NGS) has been reported to detect fusion transcripts in some malignancies. Therefore, the present study aimed to evaluate the utility of the targeted NGS in detecting the COL1A1–PDGFB fusion in patients with DFSP. MethodsWe designed a targeted DNA capture panel to tile along the fusion regions, including exon, intron, and untranslated regions of the COL1A1 and PDGFB. A cohort of 18 DNA samples extracted from formalin-fixed, paraffin-embedded tissues was used to evaluate the targeted NGS. The results were compared with that of fluorescence in situ hybridization (FISH). ResultsThe COL1A1–PDGFB fusion was identified in 13 of 18 cases (72.2%) by targeted NGS assay. PDGFB breakpoints were constantly found in exon 2, while breakpoints in COL1A1 varied from exon 15 to 46. Of these 18 cases assayed by FISH, 12 (66.7%) exhibited COL1A1–PDGFB fusion signals. One case (P9), which was FISH-negative, was demonstrated with the fusion by targeted NGS and validated by PCR and Sanger sequencing. The targeted NGS results showed a high concordance with the results of the FISH assay (94.4%). ConclusionOur study reported a targeted NGS assay for detecting the breakpoints of the COL1A1–PDGFB fusion gene, which can be implemented in diagnosing patients with DFSP.

Conserved and context-dependent roles for pdgfrb signaling during zebrafish vascular mural cell development

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.

Modest Gains After an 8-Week Exercise Program Correlate With Reductions in Non-traditional Markers of Cardiovascular Risk.

Background: Although engaging in physical exercise has been shown to reduce the incidence of cardiovascular events, the molecular mechanisms by which exercise mediates these benefits remain unclear. Based on epidemiological evidence, reductions in traditional risk factors only accounts for 50% of the protective effects of exercise, leaving the remaining mechanisms unexplained. The objective of this study was to determine whether engaging in a regular exercise program in a real world clinical setting mediates cardiovascular protection via modulation of non-traditional risk factors, such as those involved in coagulation, inflammation and metabolic regulation.Methods and Results: We performed a prospective, cohort study in 52 sedentary patients with cardiovascular disease or cardiovascular risk factors at two tertiary medical centers between January 1, 2016 and December 31, 2019. Prior to and at the completion of an 8-week exercise program, we collected information on traditional cardiovascular risk factors, exercise capacity, and physical activity and performed plasma analysis to measure levels of fibrinolytic, inflammatory and metabolic biomarkers to assess changes in non-traditional cardiovascular risk factors. The median weight change, improvement in physical fitness, and change in physical activity for the entire cohort were: −4.6 pounds (IQR: +2 pounds, −11.8 pounds), 0.37 METs (IQR: −0.076 METs, 1.06 METs), and 252.7 kcals/week (IQR: −119, 921.2 kcals/week). In addition to improvement in blood pressure and cholesterol, patients who lost at least 5 pounds, expended at least 1,000 additional kcals/week, and/or achieved ≥0.5 MET increase in fitness had a significant reduction in plasminogen activator inhibitor-1 [9.07 ng/mL (95% CI: 2.78–15.35 ng/mL); P = 0.026], platelet derived growth factor beta [376.077 pg/mL (95% CI: 44.69–707.46 pg/mL); P = 0.026); and angiopoietin-1 [(1104.11 pg/mL (95% CI: 2.92–2205.30 pg/mL); P = 0.049)].Conclusion: Modest improvements in physical fitness, physical activity, and/or weight loss through a short-term exercise program was associated with decreased plasma levels of plasminogen activator inhibitor, platelet derived growth factor beta, and angiopoietin, which have been associated with impaired fibrinolysis and inflammation.