PDGFRβ Expression Research Articles

Reversing Pdgfrβ signaling restores metabolically active beige adipocytes by alleviating ILC2 suppression in aged and obese mice

ObjectivePlatelet Derived Growth Factor Receptor Beta (Pdgfrβ) suppresses the formation of cold temperature-induced beige adipocytes in aged mammals. We aimed to determine if deleting Pdgfrβ in aged mice could rejuvenate metabolically active beige adipocytes by activating group 2 innate lymphoid cells (ILC2), and whether this effect could counteract diet-induced obesity-associated beige fat decline. MethodsWe employed Pdgfrβ gain-of-function and loss-of-function mouse models targeting beige adipocyte progenitor cells (APCs). Our approach included cold exposure, metabolic cage analysis, and age and diet-induced obesity models to examine beige fat development and metabolic function under varied Pdgfrβ activity. ResultsAcute cold exposure alone enhanced metabolic benefits in aged mice, irrespective of beige fat generation. However, Pdgfrβ deletion in aged mice reestablished the formation of metabolically functional beige adipocytes, enhancing metabolism. Conversely, constitutive Pdgfrβ activation in young mice stymied beige fat development. Mechanistically, Pdgfrβ deletion upregulated IL-33, promoting ILC2 recruitment and activation, whereas Pdgfrβ activation reduced IL-33 levels and suppressed ILC2 activity. Notably, diet-induced obesity markedly increased Pdgfrβ expression and Stat1 signaling, which inhibited IL-33 induction and ILC2 activation. Genetic deletion of Pdgfrβ restored beige fat formation in obese mice, improving whole-body metabolism. ConclusionsThis study reveals that cold temperature exposure alone can trigger metabolic activation in aged mammals. However, reversing Pdgfrβ signaling in aged and obese mice not only restores beige fat formation but also renews metabolic function and enhances the immunological environment of white adipose tissue (WAT). These findings highlight Pdgfrβ as a crucial target for therapeutic strategies aimed at combating age- and obesity-related metabolic decline.

SNHG14 promotes triple-negative breast cancer cell proliferation, invasion, and chemoresistance by regulating the ERK/MAPK signaling pathway.

The functional role and molecular mechanisms of small-nucleolar RNA host gene 14 (SNHG14) in triple-negative breast cancer (TNBC) progression remain unclear. The expression levels of SNHG14 in breast cancer samples and cell lines were determined using real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion abilities were detected using MTS and transwell assays. By RNA sequencing, differentially expressed genes were identified between the SNHG14 siRNA and the negative control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to predict the targets and pathways regulated by SNHG14. pRAF, pMEK, and pERK expression were measured by western blot. The xenograft model was constructed to access the biological function of SNHG14 in vivo. A minimal patient-derived xenograft model was established to evaluate the sensitivity to chemotherapy drugs. Our data indicated that SNHG14 expression was increased in TNBC tissues and cell lines. SNHG14 knockdown attenuated the proliferation, migration, and invasion abilities of TNBC cells both in vivo and in vitro. High SNHG14 expression was associated with lymph node metastasis and a high Ki67 index. The targets of SNHG14 were mainly enriched in the MAPK signaling pathway. pRAF, pMEK, and pERK expression were downregulated after being transfected with SNHG14 siRNA. Compared with the negative control group, the expression of CACNA1I, DUSP8, FGF17, FGFR4, FOS, PDGFRB, and DDIT3 was increased, and the expression of MKNK1 was decreased in the SNHG14 siRNA group. Minimal patient-derived xenograft model demonstrated that knockdown of SNHG14 enhanced the sensitivity to Docetaxel in vivo. Compared with the DMSO group, the proliferation of Docetaxel-resistant MDA-MB-231 cells was decreased in Dabrafenib, PD184352, and FR180204 treatment groups. SNHG14 knockdown inhibits TNBC progression by regulating the ERK/MAPK signaling pathway, which provides evidence for SNHG14 as a potential target for TNBC therapy.

Biophysically stressed vascular smooth muscle cells express MCP-1 via a PDGFR-β-HMGB1 signaling pathway.

Vascular smooth muscle cells (VSMCs) under biophysical stress play an active role in the progression of vascular inflammation, but the precise mechanisms are unclear. This study examined the cellular expression of monocyte chemoattractant protein 1 (MCP-1) and its related mechanisms using cultured rat aortic VSMCs stimulated with mechanical stretch (MS, equibiaxial cyclic stretch, 60 cycles/ min). When the cells were stimulated with 10% MS, MCP-1 expression was markedly increased compared to those in the cells stimulated with low MS intensity (3% or 5%). An enzyme-linked immunosorbent assay revealed an increase in HMGB1 released into culture media from the cells stimulated with 10% MS compared to those stimulated with 3% MS. A pretreatment with glycyrrhizin, a HMGB1 inhibitor, resulted in the marked attenuation of MCP-1 expression in the cells stimulated with 10% MS, suggesting a key role of HMGB1 on MCP-1 expression. Western blot analysis revealed higher PDGFR-α and PDGFR-β expression in the cells stimulated with 10% MS than 3% MS-stimulated cells. In the cells deficient of PDGFR-β using siRNA, but not PDGFR-α, HMGB1 released into culture media was significantly attenuated in the 10% MS-stimulated cells. Similarly, MCP-1 expression induced in 10% MS-stimulated cells was also attenuated in cells deficient of PDGFR-β. Overall, the PDGFR-β signaling plays a pivotal role in the increased expression of MCP-1 in VSMCs stressed with 10% MS. Therefore, targeting PDGFR-β signaling in VSMCs might be a promising therapeutic strategy for vascular complications in the vasculatures under excessive biophysical stress.

Galectin-1 Attenuates PDGF-Mediated AKT Signaling in Retinal Pigment Epithelial Cells.

Galectins have the potential to interact with transmembrane glycoproteins to modulate their functions. Since galectin-1 interacts with PDGF-Rβ, we analyzed the effect of galectin-1 on PDGF-BB-mediated AKT signaling in primary human retinal pigment epithelial (RPE) cells and galectin-1-deficient immortalized human RPE cells (LGALS1-/-/ARPE-19) following incubation with PDGF-BB and galectin-1. Expression and localization of galectin-1, PDGF-Rβ and pAKT were investigated using western blot analysis and immunohistochemical staining. Cell proliferation of RPE cells was analyzed using BrdU ELISA. Following treatment of human RPE cells with human recombinant (hr)-galectin-1 and PDGF-BB, an intense clustering of PDGF-Rβ and colocalization with galectin-1 were detected. By Western blot analysis and immunocytochemistry of human RPE cells, an enhanced PDGF-BB-mediated expression of pAKT was observed, which was substantially reduced by additional incubation with hr-galectin-1. Vice versa, in LGALS1-/-/ARPE-19 cells, the PDGF-BB-induced pAKT signal was enhanced compared to wild-type cells. Furthermore, a decreased expression of PDGF-Rβ in human RPE cells was observed after treatment with PDGF-BB and hr-galectin-1, while in untreated LGALS1-/-/ARPE-19 cells, its constitutive expression was increased. In addition, after treatment of RPE cells with hr-galectin-1, the PDGF-BB-induced proliferation was markedly reduced. In summary, galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in human RPE cells-an effect that is most likely facilitated via a decreased expression of PDGF-Rβ.

Calpeptin improves the cognitive function in Alzheimer's disease-like complications of diabetes mellitus rats by regulating TXNIP/NLRP3 inflammasome.

Diabetes mellitus (DM) is closely associated with Alzheimer's disease (AD), and is considered an accelerator of AD. Our previous study has confirmed that the Calpain inhibitor Calpeptin may alleviate AD-like complications of diabetes mellitus. This work further investigated its underlying mechanism. Diabetes mellitus rat model was constructed by a high-fat and high-sugar diet combined with streptozotocin, followed by the administration of Calpeptin. Moreover, rats were micro-injected with LV-TXNIP-OE/vector into the CA1 region of the hippocampus one day before streptozotocin injection. The Morris water maze test assessed the spatial learning and memory ability of rats. Immunohistochemistry and western blotting detected the expression of the pericyte marker PDGFRβ, tight junction proteins occludin and ZO-1, calpain-1, calpain-2, APP, Aβ, Aβ-related, and TXNIP/NLRP3 inflammasome-related proteins. Immunofluorescence staining examined the blood vessel density and neurons in the hippocampus. Evans blue extravasation and fluorescence detected the permeability of the blood-brain barrier (BBB) in rats. Additionally, the oxidative stress markers and inflammatory-related factors were assessed by enzyme-linked immunosorbent assay. Calpeptin effectively reduced the expression of Calpain-2 and TXNIP/NLRP3 inflammasome-related proteins, improved the decreased pericyte marker (PDGFR-β) and cognitive impairment in hippocampus of DM rats. The neuronal loss, microvessel density, permeability of BBB, Aβ accumulation, inflammation, and oxidative stress injury in the hippocampus of DM rats were also partly rescued by calpeptin treatment. The influence conferred by calpeptin treatment was reversed by TXNIP overexpression. These data demonstrated that calpeptin treatment alleviated AD-like symptoms in DM rats through regulating TXNIP/NLRP3 inflammasome. Thus, calpeptin may be a potential drug to treat AD-like complications of diabetes mellitus.

Dynamics of the thymic transcriptome at stages of acute thymic involution in Japanese Black calves with a poor prognosis

Transcriptome analysis was performed on the thymus of Japanese Black calves that were necropsied due to poor prognosis, to characterize changes associated with acute thymic involution. Gene expression profiles obtained by DNA microarray analysis of eight calf thymuses were classified into three patterns that correlated with the histopathological stage of acute thymic involution. Using principal component analysis, the first principal component of the global gene expression levels in the calf thymus was associated with the stage of acute thymic involution, suggesting that histopathological changes greatly influence the gene expression profile. Gene ontology enrichment analysis revealed that genes related to cell proliferation, wound healing, and inflammatory responses were the main contributors to the first principal component. Real-time RT-PCR showed that the thymus had lower expression of PCNA, KIFC1, and HES6, and higher expression of SYNPO2, PDGFRB, and TWIST1 during acute thymic involution. Immunohistochemistry demonstrated a decrease in the rate of Ki67-positive cells in the thymic cortex during the late stage of acute thymic involution. The rate of cleaved caspase-1-positive cells increased in the thymic cortex at an earlier stage than the increase in the rate of cleaved caspase-3-positive cells. Vimentin, which was almost absent in the non-involuted thymic cortex, appeared in the thymic cortex during acute thymic involution. These results suggest that in farmed calves with a poor prognosis, inflammatory responses and impaired thymocyte proliferation are primarily involved in acute thymic involution.

RAGE mediates hippocampal pericyte responses and neurovascular unit lesions after TBI

Traumatic brain injury impairs brain function through various mechanisms. Recent studies have shown that alterations in pericytes in various diseases affect neurovascular function, but the effects of TBI on hippocampal pericytes remain unclear. Here, we investigated the effects of RAGE activation on pericytes after TBI using male C57BL/6 J mice. Hippocampal samples were collected at different time points within 7 days after TBI, the expression of PDGFR-β, NG2 and the HMGB1-S100B/RAGE signaling pathway was assessed by Western blotting, and the integrity of the hippocampal BBB at different time points was measured by immunofluorescence. RAGE-associated BBB damage in hippocampal pericytes occurred early after cortical impact. By culturing primary mouse brain microvascular pericytes, we determined the different effects of HMGB1-S100B on pericyte RAGE. To investigate whether RAGE blockade could protect neurological function after TBI, we reproduced the process of CCI by administering FPS-ZM1 to RAGE−/− mice. TEM images and BBB damage-related assays showed that inhibition of RAGE resulted in a significant improvement in the number of hippocampal vascular basement membranes and tight junctions and a reduction in perivascular oedema compared with those in the untreated group. In contrast, mouse behavioural testing and doublecortin staining indicated that targeting the HMGB1-S100B/RAGE axis after CCI could protect neurological function by reducing pericyte-associated BBB damage. In conclusion, the present study provides experimental evidence for the strong correlation between the pericyte HMGB1-S100B/RAGE axis and NVU damage in the hippocampus at the early stage of TBI and further demonstrates that pericyte RAGE serves as an important target for the protection of neurological function after TBI.

P-209 Development and characterization of human uterine myometrium in prenatal stage: insights from a single-cell atlas and three-dimensional imaging

Abstract Study question What’s the timepoint of human uterine myometrial formation and how dose it develop in the prenatal stage. Summary answer This study identified that the myometrium layer initially forms at postconceptional week (PCW) 11, ESR1+, IGFBP5, POSTIN may play pivotal role in myometrial development. What is known already Using immunohistochemistry staining, previous studies observed a faint expression of α-SMA in the outer mesenchyme at approximately 8-9 weeks of gestation and showed the early development of muscular layer in the uterine wall, and the expression of α-SMA becomes more prominent in the uterine corpus and cervix by 18 weeks, suggesting the maturation of smooth muscle cells may commence from 18 weeks of gestation. ESR-1 gene encodes estrogen receptor alpha, which plays a pivotal role in menstruation, pregnancy, and parturition. In adult female myometrium, estrogen performs functions in proliferation before term and contractile at term. Study design, size, duration This is an observational study. To investigate when exactly the smooth-muscle tissue appears during uterine development, we have collected 14 uterine tissue samples from 14 aborted fetuses across seven gestational time points from PCW 8 to PCW 15. Participants/materials, setting, methods Uterus from aborted fetuses from PCW8- PCW15 cover the fusion of Mullerian tube and development of Myometrium. The 10X genomics scRNA-seq and immune labeling were performed. In addition, we have integrated scRNA-seq data from adult uterine tissue for comparison. Main results and the role of chance We provide a single cell atlas of the human developing uterus, and we observed that smooth-muscle cells (SMCs), identified by ACTA2, TAGLN, MYH11, exhibited a significant increase in abundance starting from PCW 11, but weaker signals of SMCs were also detected at earlier stages. Using the iDISCO technique with continuous α-SMA labeling to examine the localization of α-SMA in the uterus between PCW10- PCW12, we observed the entire process of myometrial development in 3-D level. We utilized an oblique section of 3-D reconstruction uterus and extended it to 150 μm. Subsequently, apparent signals became visible in the middle of the uterine wall and the uterine fundus. Collectively, these findings suggest that the anatomical myometrium layer initially forms at PCW 11. We used the CytoTRACE to assess the capacity of differentiation of adult and fetal SMCs, SMCs come from fetus appeal a significant grander potential. Specifically, fetal uterine SMCs showed higher expression of ESR1, IGFBP5, and POSTN, while adult uterine SMCs exhibited higher expression of PDGFRB and IGFBP7. These results suggested ESR1+ SMCs could be the precursor of SMCs and SMCs in fetal uterus could be in a primary state. Limitations, reasons for caution While our study did not capture later stages of development or postnatal changes. Future studies are needed to understand the full spectrum of myometrial development. It is also important to note that the findings in this study are specific to human and may not directly translate to other species. Wider implications of the findings Our study provides a basis for studying abnormal myometrial development and its association with various reproductive disorders, such as the abnormal uterine development in female fetus with Turner syndrome. Potential biomarkers or therapeutic targets for uterine malformations and abnormal uterine contractility may also of research interest. Trial registration number not applicable

Reversing Pdgfrβ Signaling Restores Metabolically Active Beige Adipocytes by Alleviating ILC2 Suppression in Aged and Obese Mice.

Platelet Derived Growth Factor Receptor Beta (Pdgfrβ) suppresses the formation of cold temperature-induced beige adipocytes in aged mammals. We aimed to determine if deleting Pdgfrβ in aged mice could rejuvenate metabolically active beige adipocytes by activating group 2 innate lymphoid cells (ILC2), and whether this effect could counteract diet-induced obesity-associated beige fat decline. We employed Pdgfrβ gain-of-function and loss-of-function mouse models targeting beige adipocyte progenitor cells (APCs). Our approach included cold exposure, metabolic cage analysis, and age and diet-induced obesity models to examine beige fat development and metabolic function under varied Pdgfrβ activity. Acute cold exposure alone enhanced metabolic benefits in aged mice, irrespective of beige fat generation. However, Pdgfrβ deletion in aged mice reestablished the formation of metabolically functional beige adipocytes, enhancing metabolism. Conversely, constitutive Pdgfrβ activation in young mice stymied beige fat development. Mechanistically, Pdgfrβ deletion upregulated IL-33, promoting ILC2 recruitment and activation, whereas Pdgfrβ activation reduced IL-33 levels and suppressed ILC2 activity. Notably, diet-induced obesity markedly increased Pdgfrβ expression and Stat1 signaling, which inhibited IL-33 induction and ILC2 activation. Genetic deletion of Pdgfrβ restored beige fat formation in obese mice, improving whole-body metabolism. This study reveals that cold temperature exposure alone can trigger metabolic activation in aged mammals. However, reversing Pdgfrβ signaling in aged and obese mice not only restores beige fat formation but also renews metabolic function and enhances the immunological environment of white adipose tissue (WAT). These findings highlight Pdgfrβ as a crucial target for therapeutic strategies aimed at combating age- and obesity-related metabolic decline.

Promotion of mature angiogenesis in ischemic stroke by Taohong Siwu decoction through glycolysis activation.

Backgrounds: Mature angiogenesis plays a critical role in improving cerebral ischemia-reperfusion injury (CIRI). Glycolysis serves as the primary energy source for brain microvascular endothelial cells (BMECs), whereas other vascular cells rely on aerobic respiration. Therefore, intercellular variations in energy metabolism could influence mature angiogenesis. Taohong Siwu Decoction (THSWD) has demonstrated efficacy in treating ischemic stroke (IS), yet its potential to promote mature angiogenesis through glycolysis activation remains unclear. Methods: In this study, we established a middle cerebral artery occlusion/reperfusion (MCAO/R) model in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. We assessed neuroprotective effects using neurobehavioral scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin-eosin (HE) staining, and Nissl staining in MCAO/R rats. Additionally, we evaluated mature angiogenesis and glycolysis levels through immunofluorescence, immunohistochemistry, and glycolysis assays. Finally, we investigated THSWD's mechanism in linking glycolysis to mature angiogenesis in OGD/R-induced BMECs. Results: In vivo experiments demonstrated that THSWD effectively mitigated cerebral damage and restored neurological function in MCAO/R rats. THSWD significantly enhanced CD31, Ang1, PDGFB, and PDGFR-β expression levels, likely associated with improved glucose, pyruvate, and ATP levels, along with reduced lactate and lactate/pyruvate ratios. In vitro findings suggested that THSWD may boost the expression of mature angiogenesis factors (VEGFA, Ang1, and PDGFB) by activating glycolysis, increasing glucose uptake and augmenting lactate, pyruvate, and ATP content, thus accelerating mature angiogenesis. Conclusion: THSWD could alleviate CIRI by activating the glycolysis pathway to promote mature angiogenesis. Targeting the glycolysis-mediated mature angiogenesis alongside THSWD therapy holds promise for IS treatment.

Mechanism of salidroside in inhibiting proliferation, migration and promoting phenotypic switching of arterial smooth muscle cells

This study aims to examine the effect of salidroside(SAL) on the phenotypic switching of human aortic smooth muscle cells(HASMC) induced by the platelet-derived growth factor-BB(PDGF-BB) and investigate the pharmacological mechanism. Firstly, the safe concentration of SAL was screened by the lactate dehydrogenase release assay. HASMC were divided into control, model, and SAL groups, and the cells in other groups except the control group were treated with PDGF-BB for the modeling of phenotypic switching. Cell proliferation and migration were detected by the cell-counting kit(CCK-8) assay and Transwell assay, respectively. The cytoskeletal structure was observed by F-actin staining with fluorescently labeled phalloidine. The protein levels of proliferating cell nuclear antigen(PCNA), migration-related protein matrix metalloprotein 9(MMP-9), fibronectin, α-smooth muscle actin(α-SMA), and osteopontin(OPN) were determined by Western blot. To further investigate the pharmacological mechanism of SAL, this study determined the expression of protein kinase B(Akt) and mammalian target of rapamycin(mTOR), as well as the upstream proteins phosphatase and tensin homologue(PTEN) and platelet-derived growth factor receptor β(PDGFR-β) and the downstream protein hypoxia-inducible factor-1α(HIF-1α) of the Akt/mTOR signaling pathway. The results showed that the HASMCs in the model group presented significantly increased proliferation and migration, the switching from a contractile phenotype to a secretory phenotype, and cytoskeletal disarrangement. Compared with the model group, SAL weakened the proliferation and migration of HASMC, promoted the expression of α-SMA(a contractile phenotype marker), inhibited the expression of OPN(a secretory phenotype marker), and repaired the cytoskeletal disarrangement. Furthermore, compared with the control group, the modeling up-regulated the levels of phosphorylated Akt and mTOR and the relative expression of PTEN, HIF-1α, and PDGFR-β. Compared with the model group, SAL down-regulated the protein levels of phosphorylated Akt and mTOR, PTEN, PDGFR-β, and HIF-1α. In conclusion, SAL exerts a protective effect on the HASMCs exposed to PDGF-BB by regulating the PDGFR-β/Akt/mTOR/HIF-1α signaling pathway.

Mutation in PDGFRbeta: a potential new pathogenic variant for acquired mitral valve prolapse

Abstract Funding Acknowledgements None. Background Mitral valve prolapse (MVP) due to myxomatous degeneration is characterized by familial clustering, but few pathogenic genes have been identified. In a genetic screening program for MVP patients, we identified a platelet-derived growth factor receptor β (PDGFRβ)-E162K missense variant in a family with a bi-leaflet MVP phenotype in four affected family members. PDGFRβ is a receptor tyrosine kinase, which plays an important role in vascular development. Purpose In this study, we investigated whether the E162K substitution in PDGFRβ could lead to mitral valve abnormalities. Methods To assess the functional effects of the missense variant, proliferation and migration assays were performed using HeLa cells overexpressing wild-type (WT) or mutant PDGFRβ. Immunostainings were performed to determine PDGFRβ expression in the diseased valves from a family member undergoing surgical operation and from additional patients operated for MVP. Mouse models were created with the mouse equivalent of the human PDGFRβ-E162K variant (PDGFRβ-E161K) using CRISPR/CAS9. The variant was not lethal and transthoracic echocardiography was performed on adult homozygous PDGFRβE161K/E161K mice, heterozygous PDGFRβE161K/- mice, and WT mice . In addition, histology was performed in adult and neonatal mice to detect potential valvular defects. Results HeLa cells expressing PDGFRβ-E162K and stimulated with PDGFD showed reduced proliferation and migration, suggesting a loss of function of the mutant receptor. PDGFRβ was expressed in human diseased mitral valves. Echocardiographic evaluation revealed a significant larger mitral valve annulus diameter in both PDGFRβE161K/- and PDGFRβE161K/E161K compared to WT (WT: 1.97 mm; PDGFRβE161K/-:2.13, p=0.031; PDGFRβE161K/E161K:2.42 mm, p<0.0001). Histological data in PDGFRβE161K/E161K mice showed significant leaflet thickening in the free edge (FE) of the posterior mitral valve leaflet (PMVL) compared to WT (WT: 72.34 ɥm; PDGFRβE161K/E161K: 109.6 ɥm, p=0.040) and a larger leaflet area of the PMVL compared to WT (WT: 25280 ɥm²; PDGFRβE161K/E161K: 44494 ɥm², p=0.020). Moreover, an altered organization of the extracellular matrix reminiscent of the myxomatous degeneration in MVP was observed. These alterations were not observed in mutant neonatal hearts. Conclusions The PDGFRβ-E162K variant is associated with familial MVP and alters the function of PDGFRβ. Mice harboring this mutation display acquired mitral valve defects.

Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-β (PDGFR-β)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-β. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-β. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

Healing activity of a muco-adhesive formula of Teucrium polium against acetic acid-induced oral ulcer in rats

Oral ulcers are highly common painful conditions that harmfully affect the quality of life and require long-term therapy. Muco-adhesive preparations provide efficient and convenient delivery systems that prolong the drug retention time necessary for the different stages of ulcer healing. Therefore, the study aimed to evaluate the healing activity of Teucrium polium in a muco-adhesive formula against acetic acid-induced oral ulcers in rats. Acetic acid was applied to the inner cheeks of the rats and the muco-adhesive formula was applied once daily for 14 days. Acetic acid was found to disrupt the oral tissue histological features, increase collagen deposition, induce oxidative stress through increasing malondialdehyde (MDA) levels and decreasing superoxide dismutase and catalase activities, increase the expression of the inflammatory biomarkers, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and decreased hydroxyproline content and the expression of the factors that contribute in wound healing including transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor receptor 1 (VEGFR1), and platelet-derived growth factor receptor beta (PDGFRβ). However, the daily application of a mucoadhesive formula impregnated with T. polium, significantly restored the regular histological features of the oral tissue, decreased the concentration of MDA, TNF-α, IL-6, and COX-2, increased superoxide dismutase and catalase activities, and restored the expression of TGF-β1, VEGFR1, and PDGFRβ. Therefore, T. polium extract muco-adhesive formula can be considered a potential therapy for chronic oral ulcers.

Exploring the impact of PDGFD in osteosarcoma metastasis through single-cell sequencing analysis

PurposeThe overall survival rate for metastatic osteosarcoma hovers around 20%. Responses to second-line chemotherapy, targeted therapies, and immunotherapies have demonstrated limited efficacy in metastatic osteosarcoma. Our objective is to validate differentially expressed genes and signaling pathways between non-metastatic and metastatic osteosarcoma, employing single-cell RNA sequencing (scRNA-seq) and additional functional investigations. We aim to enhance comprehension of metastatic mechanisms and potentially unveil a therapeutic target.MethodsscRNA-seq was performed on two primary osteosarcoma lesions (1 non-metastatic and 1 metastatic). Seurat package facilitated dimensionality reduction and cluster identification. Copy number variation (CNV) was predicted using InferCNV. CellChat characterized ligand-receptor-based intercellular communication networks. Differentially expressed genes underwent GO function enrichment analysis and GSEA. Validation was achieved through the GSE152048 dataset, which identified PDGFD-PDGFRB as a common ligand-receptor pair with significant contribution. Immunohistochemistry assessed PDGFD and PDGFRB expression, while multicolor immunofluorescence and flow cytometry provided insight into spatial relationships and the tumor immune microenvironment. Kaplan-Meier survival analysis compared metastasis-free survival and overall survival between high and low levels of PDGFD and PDGFRB. Manipulation of PDGFD expression in primary osteosarcoma cells examined invasion abilities and related markers.ResultsTen clusters encompassing osteoblasts, osteoclasts, osteocytes, fibroblasts, pericytes, endothelial cells, myeloid cells, T cells, B cells, and proliferating cells were identified. Osteoblasts, osteoclasts, and osteocytes exhibited heightened CNV levels. Ligand-receptor-based communication networks exposed significant fibroblast crosstalk with other cell types, and the PDGF signaling pathway was activated in non-metastatic osteosarcoma primary lesion. These results were corroborated by the GSE152048 dataset, confirming the prominence of PDGFD-PDGFRB as a common ligand-receptor pair. Immunohistochemistry demonstrated considerably greater PDGFD expression in non-metastatic osteosarcoma tissues and organoids, correlating with extended metastasis-free and overall survival. PDGFRB expression showed no significant variation between non-metastatic and metastatic osteosarcoma, nor strong correlations with survival times. Multicolor immunofluorescence suggested co-localization of PDGFD with PDGFRB. Flow cytometry unveiled a highly immunosuppressive microenvironment in metastatic osteosarcoma. Manipulating PDGFD expression demonstrated altered invasive abilities and marker expressions in primary osteosarcoma cells from both non-metastatic and metastatic lesions.ConclusionsscRNA-seq illuminated the activation of the PDGF signaling pathway in primary lesion of non-metastatic osteosarcoma. PDGFD displayed an inhibitory effect on osteosarcoma metastasis, likely through the suppression of the EMT signaling pathway.

Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. Cancer-associated fibroblasts (CAFs) promote progression of cancer, but their role in cSCC is largely unknown. We examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. We utilized multiplexed fluorescence immunohistochemistry for profiling CAF landscape in metastatic and non-metastatic primary human cSCCs, in metastases, and in premalignant epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and results were correlated with clinical and histopathological parameters including disease-specific mortality. Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression to invasive cSCC. Elevation of FAP and platelet-derived growth factor receptor-β (PDGFRβ) expression was associated with metastasis risk of primary cSCCs. High expression of PDGFRβ and periostin correlated with poor prognosis. Multimarker combination defined CAF subset, PDGFRα-/PDGFRβ+/FAP+, was associated with invasion and metastasis, and independently predicted poor disease-specific survival. These results identify high PDGFRβ expression alone and multimarker combination PDGFRα-/PDGFRβ+/FAP+ by CAFs as potential biomarkers for risk of metastasis and poor prognosis.

In vitro modeling of recurrent Dermatofibrosarcoma Protuberans: Assessment of 5-aminolevulinic acid photodynamic therapy efficacy

BackgroundDermatofibrosarcoma Protuberans (DFSP) is a rare, low-grade malignant tumor of the dermis with a high recurrence rate post-surgery. Current treatments, including surgery, radiotherapy, and targeted therapy, have limitations. Photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA) is a promising non-invasive approach, but its efficacy in DFSP treatment remains underexplored. MethodsThis study aimed to evaluate the anti-tumor efficacy of 5-ALA PDT using an in vitro model derived from a recurrent DFSP patient. The cells were treated with varying concentrations of 5-ALA and exposed to red light, followed by assessments of cell viability, proliferation, apoptosis, migration, invasion, angiogenesis, and expression of DFSP-related genes and proteins. Results5-ALA PDT significantly reduced DFSP cell viability in a dose-dependent manner and induced apoptosis. It also effectively inhibited cell proliferation, migration, and invasion, as well as suppressed angiogenic activity in conditioned media. Furthermore, 5-ALA PDT downregulated the expression of COL1A1 and PDGFRB, key genes in DFSP pathogenesis. ConclusionsThe findings provide the first evidence of 5-ALA PDT's in vitro anti-tumor efficacy against DFSP, suggesting its potential as a novel therapeutic approach for DFSP. Further studies are warranted to explore the clinical utility of 5-ALA PDT in preventing DFSP recurrence.

Sinomenine inhibits PDGF/PDGFR signaling pathway to reduce RA-FLS migration induced by NETs

This study aims to decipher the mechanism of sinomenine in inhibiting platelet-derived growth factor/platelet-derived growth factor receptor(PDGF/PDGFR) signaling pathway in rheumatoid arthritis-fibroblast-like synoviocyte(RA-FLS) migration induced by neutrophil extracellular traps(NETs). RA-FLS was isolated from the synovial tissue of 3 RA patients and cultured. NETs were extracted from the peripheral venous blood of 4 RA patients and 4 healthy control(HC). RA-FLS was classified into control group, HC-NETs group, RA-NETs group, RA-NETs+sinomenine group and RA-NETs+sinomenine+CP-673451 group. RNA-sequencing(RNA-seq) was conducted to identify the differentially expressed genes between HC-NETs and RA-NETs groups. Sangerbox was used to perform the Gene Ontology(GO) function and the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment. Cytoscape was employed to build the protein-protein interaction(PPI) network. AutoDock Vina and PyMOL were used for molecular docking of sinomenine with PDGFβ and PDGFRβ. The cell proliferation and migration were determined by the cell counting kit-8(CCK-8) and cell scratch assay, respectively. Western blot was employed to determine the protein level of PDGFRβ. Real-time quantitative polymerase chain reaction(RT-qPCR) was carried out to determine the mRNA levels of matrix metalloproteinases(MMPs). The results revealed that neutrophils in RA patients were more likely to produce NETs. Compared with HC-NETs group, RA-NETs group showed up-regulated expression of PDGFβ and PDGFRβ. Compared with control group, RA-NETs group showed increased cell proliferation and migration and up-regulated protein level of PDGFRβ and mRNA levels of PDGFβ, PDGFRβ, MMP1, MMP3, and MMP9(P<0.05). Compared with RA-NETs group, RA-NETs+sinomenine group presented decreased cell proliferation and migration and down-regulated protein and mRNA level of PDGFRβ and mRNA levels of MMP1, MMP3, and MMP9(P<0.05). Compared with RA-NETs+sinomenine group, the proliferation ability of RA-NETs+sinomenine+CP-673451 group decreased(P<0.05). The findings prove that sinomenine reduces the RA-NETs-induced RA-FLS migration by inhibiting PDGF/PDGFR signaling pathway, thus mitigating RA.

Abstract 291: Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types

Abstract Background: Stromal cells, including CAFs, are a heterogeneous group of cells in the tumor microenvironment. Many models describe their heterogeneity like the classification into 3 main subtypes in breast cancer: myCAF (myofibroblasts), iCAF (inflammatory CAF), and PVL (perivascular-like) cells. However, a pan-cancer model has not been established to exhaustively characterize stromal cell heterogeneity, undermining efforts in targeting CAFs with anti-tumor therapies. We investigated the heterogeneity of stromal cells in diverse diagnoses with single-cell RNA sequencing (scRNA-seq) data and bulk RNA-seq of sorted stromal cells to define consistent subtypes. Design: First, a meta-cohort of 11 scRNA-seq open-source datasets (>500,000 cells, >100 patients, 6 diagnoses) was analyzed. We also generated a scRNA-seq dataset of CD90+ cells derived from tumor tissue of an ovarian cancer patient. Additionally, bulk RNA-seq of 6 PVL, 2 iCAF, and 4 myCAF sorted cell samples across four solid cancers was performed. scRNA-seq data was analyzed with the SCANPY Python package. Results: Stromal cell clusters were defined as positive for PDGFRB, THY1 (CD90), and COL1A1 expression, and negative for the expression of known non-stromal and endothelial cell markers, by separately analyzing the open-source scRNA-seq datasets. Analysis of stromal cell heterogeneity revealed that the CAFs were consistently classified into 3 subtypes across all datasets: myCAF (high expression: FAP and ACTA2), PVL (high expression: MCAM (CD146) and ACTA2), and iCAF (high expression: APOD and cytokines IL6, CXCL12, CXCL2). Differential gene expression analysis of the scRNA-seq meta-cohort allowed us to develop functional gene expression signatures to define each subtype. All scRNA-sequenced CD90+ cells from ovarian cancer tissue were classified into the CAF subtypes. The myCAF and PVL signatures were specifically upregulated in the corresponding cell types (p<0.01, Mann-Whitney U test). While the iCAF signature was enriched in both iCAFs and myCAFs, myCAF and PVL signatures were downregulated in iCAFs. Finally, we developed a fluorescence-activated cell sorting (FACS) panel to separate CAF subtypes, using negative markers (CD45, CD31, EPCAM), a positive marker for stromal cells (CD90) and cell-type surface markers (CD146, FAP, CD91, CD44) for myCAF, iCAF and PVL cells. The high expression of CAF-specific genes in the sorted CAF populations denoted high sample purity. Importantly, the 3 CAF subpopulations separated by gating based on different levels of cell-type surface markers showed expression patterns congruent with the developed signatures across diagnoses. Summary: Altogether, the consistent classification of stromal cells with our cell-specific expression profiles demonstrated that the 3 CAF subtypes are conserved in many cancer types, offering groundwork for a pan-cancer model of stromal cell heterogeneity. Citation Format: Andrey Kravets, Boris Shpak, Anastasia Zotova, Maria Savchenko, Sofya Kust, Olga Golubeva, Mary Abdou, Fedor Grigoryev, Irina Bulusheva, Beniamin Sargsyan, Polina Pavlovich, Vlad Maximov, Vladimir Kushnarev, Michael Goldberg, Nikita Kotlov, Alexander Bagaev. Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types [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 291.

Linc01124 promotes hepatoblastoma proliferation through the miR-24-3p/PI3K/AKT pathway

Children diagnosed with advanced hepatoblastoma (HB) generally have a poor prognosis and are in urgent need of effective treatment strategies. Long non-coding RNAs (lncRNAs) have been found to play crucial roles in various malignant tumors and can be potential targets for tumor diagnosis, treatment and prognosis. However, the role of most lncRNAs in HB pathogenesis is still not well understood. In this study, we investigated the expression of Linc01124 and its effect in HB. Clinical samples of HB patients were collected, and differentially expressed lncRNAs in HB were screened by quantitative real-time polymerase chain reaction (qRT-PCR). The Cell Counting kit-8 and 5-ethynyl-2′-deoxyuridine assay were used to detect cell proliferation in HB cells. The HB cells' apoptosis was assessed by the flow cytometry and terminaldeoxynucleotidyl transferase-mediated nick-end labeling assay. Then micro-RNAs and messenger RNAs targeted by Linc01124 were selected according to miRanda and miRTarbase. The gene pathways regulated by Linc01124 were predicted through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. The prediction results were subsequently validated using qRT-PCR and Western blotting (WB). A Pearson's correlation analysis was conducted to examine the correlation between hsa-miR-24-3p and Linc01124, IL4, PDGFRB, BRCA1, and NOS3. In addition, HB cells were treated with a Linc01124 inhibitor and PI3K-Akt pathway activator to evaluate the involvement of miR-24-3p/PI3K/AKT in the regulation of HB cell function by Linc01124. Linc01124 was up-regulated in HB cells and tissues. Knockdown of Linc01124 can reduce cell proliferation and increase apoptosis. Bioinformatics analysis predicted that hsa-miR-24-3p could target Linc01124 and 8 genes involved in the PI3K-Akt pathway. qRT-PCR and WB experiments also demonstrated that disrupting Linc01124 expression could increase miR-24-3p expression and inhibit the PI3K/AKT pathway. Meanwhile, the target genes IL4, PDGFRB, BRCA1 and NOS3 expression were also down-regulated by silencing Linc01124. Hsa-miR-24-3p was found to have a negative association with Linc01124, IL4, PDGFRB, BRCA1 and NOS3. PI3K/AKT activator could rescue the decreased proliferation and increased apoptosis induced by Linc01124 knockdown. Linc01124 plays an oncogenic role by improving the activity of the PI3K/AKT pathway, which provides a theoretical basis for future research on the pathogenesis and progression mechanism of HB.