Mitogen For Smooth Muscle Cells Research Articles

Abstract TMP112: Von Willebrand Factor (VWF) Modulates Smooth Muscle Cell Proliferation During the Outward Remodeling of Leptomeningeal Collateral Arterioles Following Stroke

Introduction: The leptomeningeal collaterals (LMCs) undergo outward remodeling after stroke to increase rescue flow toward the occluded region. We previously showed increased VWF in remodeled LMCs and others have shown that VWF can act as a smooth muscle cell (SMC) mitogen. Here we test the hypothesis that VWF modulates vascular remodeling of LMCs in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos), aged (cohort 2; 24 mos) or VWF KO and WT male mice (cohort 3; 4 mos). At 3, 7, and 14 days after pdMCAO, the pial vessels were processed for VWF/Ki67/SMA immunofluorescence and morphometric analysis. Lastly, distribution of VWF was evaluated in LMCs from human and pig brains. Results: During the 3-14 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF in ipsilateral LMCs as well as increased Ki67 positive cells. In ECs, Ki67-positive cells were 10.68 ± 1.48 and 10.73 ± 1.26 per mm of vessel at 3 and 7 days, respectively. In SMCs, Ki67-positive cells were 0.049 ± 0.22 and 4.546 ± 0.95 per mm vessel at 3 and 7 days, respectively. By 14 days, LMC no longer showed Ki67-positive ECs or SMCs. In aged cohort 2, along with attenuated VWF expression and remodeling, the Ki67-positive ECs and SMCs were significantly decreased in the aged mice compared to young mice (Ki67-positive ECs were reduced by 40% and 50% at 3 and 7 days, respectively; Ki67-positive SMCs were reduced by 75% at 7 days). In cohort 3, Ki67-positive SMCs were absent in the VWF-KOs at 3 days post-stroke. However, by 7 days, Ki67-positive SMCs were equivalent between WT and VWF-KOs (4.8-5.0 per mm vessel). Lastly, the presence of VWF was detected in the smooth muscle layer of LMCs of human and pig brains. Conclusions: Our study revealed increased VWF in the mural layer of remodeled LMCs and both EC and SMC proliferation. The remodeling response, VWF expression, and number of proliferative cells were attenuated in LMCs from aged brains. Knockout of VWF resulted in reduced LMC remodeling and delayed SMC proliferation. Our findings also provide evidence for VWF within the smooth muscle layer of pig and human brain arterioles, supporting the broader potential role of VWF as a molecular player in vascular remodeling.

Exposure to real-ambient particulate matter induced vascular hypertrophy through activation of PDGFRβ

BackgroundVascular toxicity induced by particulate matter (PM) exposure exacerbates the onset and development of cardiovascular diseases; however, its detailed mechanism remains unclear. Platelet-derived growth factor receptor β (PDGFRβ) acts as a mitogen for vascular smooth muscle cells (VSMCs) and is therefore essential for normal vasoformation. However, the potential effects of PDGFRβ on VSMCs in PM-induced vascular toxicity have not yet been elucidated. MethodsTo reveal the potential roles of PDGFRβ signalling in vascular toxicity, individually ventilated cage (IVC)-based real-ambient PM exposure system mouse models and PDGFRβ overexpression mouse models were established in vivo, along with in vitro VSMCs models. ResultsVascular hypertrophy was observed following PM-induced PDGFRβ activation in C57/B6 mice, and the regulation of hypertrophy-related genes led to vascular wall thickening. Enhanced PDGFRβ expression in VSMCs aggravated PM-induced smooth muscle hypertrophy, which was attenuated by inhibiting the PDGFRβ and janus kinase 2 /signal transducer and activator of transcription 3 (JAK2/STAT3) pathways. ConclusionOur study identified the PDGFRβ gene as a potential biomarker of PM-induced vascular toxicity. PDGFRβ induced hypertrophic effects through the activation of the JAK2/STAT3 pathway, which may be a biological target for the vascular toxic effects caused by PM exposure.

Abstract 13756: The Hematopoietic and Megakaryocytic Protective Effect of PDGF-BB on Radiation-Induced Myelosuppression Model

Introduction: Platelet-derived growth factors (PDGF) was initially purified from platelets and characterized as a mitogen for fibroblasts and smooth muscle cells. The PDGF family comprises four different genes that form at least five different dimers, including PDGF-AA, AB, BB, CC and DD. In the hematopoietic system, genetic studies have reported that knock-out mice for PDGF-BB show anemia and thrombocytopenia. PDGF also stimulates the proliferation of megakaryocytes, erythrocytes, leukocytes, and their progenitors, and enhances the expansion of megakaryocyte progenitors from human CD34+ cells. Methods: Blood counts, bone marrow histology, and CFU were performed to investigate the protective effect of PDGF-BB on mice bone marrow hematopoiesis. Flow cytometry was used to detect early apoptosis (Annexin V), mitochondrial membrane potential (JC-1) and Caspase-3 of Meg-01. Results: Peripheral blood count showed that PDGF stimulated the recovery of white blood cells and platelets on radiation-induced myelosuppression model (P<0.01, n=4). Bone marrow histology showed that the numbers of megakaryocytes and their progenitor cells were higher than that in PDGF-BB group. PDGF-BB significantly stimulated the formation of CFU-MK, CFU-GM, BFU-E and CFU-F (P<0.01, n=4). PDGF-BB had a dose-dependent effect on cell proliferation in the range of 5-50 μg/mL in vitro (P<0.001, n=4). Late apoptotic and necrotic cells (Annexin V +, PI +) and total dead cells (Annexin V +) were decreased in the PDGF-BB (50 μg/ml) group (P<0.01, n=4). The mitochondrial membrane potential in the PDGF-BB group was increased when compared with control group, which indicated that the number of apoptotic cells was decreased (P<0.01, n=4). Besides, the expression of Caspase-3 in PDGF-BB treatment group was also lower than that in control group (P<0.05, n=4). Conclusions: PDGF-BB has a hematopoietic protective effect on the myelopoietic mice model, especially on megakaryocytes and their progenitor cells. It has proliferative and anti-apoptotic effects on Meg-01, and the mechanism may be mediated through JC-1 and Caspase-3 pathway.

Role of cyclic AMP response element binding protein (CREB) in angiotensin II-induced responses in vascular smooth muscle cells.

Cyclic AMP response element (CRE) binding protein (CREB) is a nuclear transcription factor that regulates the transcription of several genes containing the CRE sites on their promoters. CREB is activated by phosphorylation on a key serine residue, Ser311, in response to a wide variety of extracellular stimuli including angiotensin II (Ang II). Ang II is an important vasoactive peptide and mitogen for vascular smooth muscle cells (VSMC) that in addition to regulating the contractile response in VSMC also plays an important role in phenotypic switch of VSMC from contractile to a synthetic state. The synthetic VSMC are known to exhibit proliferative and migratory properties due to hyperactivation of Ang II-induced signaling events. Ang II has been shown to induce CREB phosphorylation/activation and transcription of genes implicated in proliferation, growth, and migration. Here, we have highlighted some key studies that have demonstrated an important role of CREB in Ang II-mediated gene transcription, proliferation, hypertrophy, and migration of VSMC.

PDGF/MEK/ERK axis represses Ca2+ clearance via decreasing the abundance of plasma membrane Ca2+ pump PMCA4 in pulmonary arterial smooth muscle cells.

Pulmonary arterial hypertension (PAH) is a rare and lethal disease characterized by vascular remodeling and vasoconstriction, which is associated with increased intracellular calcium ion concentration ([Ca2+]i). Platelet-derived growth factor-BB (PDGF-BB) is the most potent mitogen for pulmonary arterial smooth muscle cells (PASMCs) and is involved in vascular remodeling during PAH development. PDGF signaling has been proved to participate in maintaining Ca2+ homeostasis of PASMCs; however, the mechanism needs to be further elucidated. Here, we illuminate that the expression of plasma membrane calcium-transporting ATPase 4 (PMCA4) was downregulated in PASMCs after PDGF-BB stimulation, which could be abolished by restraining the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK). Functionally, suppression of PMCA4 attenuated the [Ca2+]i clearance in PASMCs after Ca2+ entry, promoting cell proliferation and elevating cell locomotion through mediating formation of focal adhesion. Additionally, the expression of PMCA4 was decreased in the pulmonary artery of monocrotaline (MCT)- or hypoxia-induced PAH rats. Moreover, knockdown of PMCA4 could increase the right ventricular systolic pressure (RVSP) and wall thickness (WT) of pulmonary artery in rats raised under normal conditions. Taken together, our findings demonstrate the importance of the PDGF/MEK/ERK/PMCA4 axis in intracellular Ca2+ homeostasis in PASMCs, indicating a functional role of PMCA4 in pulmonary arterial remodeling and PAH development.

Vaginal Bromocriptine Improves Pain, Menstrual Bleeding, and Quality of Life in Women With Adenomyosis: A Pilot Study

(Abstracted from Acta Obstet Gynecol Scand 2019;98:1341–1350) Animal models suggest that increased uterine prolactin concentration is a risk factor for adenomyosis. Prolactin is produced in the human endometrium, myometrium, and the pituitary gland and acts as a smooth muscle cell mitogen in vitro.

A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.

Vaginal bromocriptine improves pain, menstrual bleeding and quality of life in women with adenomyosis: A pilot study.

Adenomyosis is a benign uterine disease where endometrial glands and stroma are found within the myometrium surrounded by an area of hypertrophic myometrium. Symptomatology includes heavy menstrual bleeding and pelvic pain. The pathogenesis of adenomyosis is not known; however, animal models have shown increased uterine concentration of prolactin as a risk factor. Prolactin acts as a smooth muscle cell mitogen. If prolactin is central to adenomyosis pathogenesis, reducing uterine prolactin could be a possible medical treatment option. In this pilot study, we aim to evaluate the effect of bromocriptine, a prolactin inhibitor, on menstrual bleeding and pain in women with adenomyosis. 23 women with diffuse adenomyosis were enrolled from a university hospital in Sweden and a tertiary care center in the USA. Nineteen patients completed 6months of treatment with vaginal bromocriptine at a dose of 5mg daily. Participants completed validated measures at baseline, 3 and 6months of treatment, and at 9months (3 months after cessation of bromocriptine). Validated measures utilized included Pictorial Blood Loss Assessment Chart (PBLAC), Aberdeen Menorrhagia Clinical Outcomes Questionnaire (AMCOQ), Visual Analog Scale for pain (VAS), McGill Pain Questionnaire (MPQ), Endometriosis Health Profile (EHP-30), Female Sexual Function Index (FSFI) and the Fibroid Symptom Quality of Life (UFS-QOL) symptom severity and health-related quality of life (HRQL) subscores. Scores were compared between baseline and 9months using the Wilcoxon signed rank test. Mean age of participants was 44.8years. About 77.8% reported PBLAC scores>250 and 68.4% reported moderate to severe pain at baseline. Compared with baseline, women had lower 9-month scores (median [interquartile range] for all) on PBLAC (baseline 349 [292-645] vs 9-month 233 [149-515], P=0.003), VAS (5.0 [4-8.3] vs 2.5 [0-4.5], P<0.001), EHP Core Pain (15.9 [9.1-50.0] vs 3.4 [2.3-34.1], P=0.029), EHP Core Self-image (41.7 [16.7-58.3] vs 25 [0-5], P=0.048) and Symptom Severity Score (60 [44-72] vs 44 [25-56], P<0.001) and higher HRQL scores (57 [37-63] vs 72 [51-85], P<0.001) following bromocriptine treatment. Other EHP core parameters and FSFI were not significantly different. Significant improvement in menstrual bleeding, pain and quality of life after vaginal bromocriptine treatment suggests a novel therapeutic agent for adenomyosis.

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

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

Hyperglycemia does not affect tissue repair responses in shear stress-induced atherosclerotic plaques in ApoE\u2212/\u2212 mice

The mechanisms responsible for macrovascular complications in diabetes remain to be fully understood. Recent studies have identified impaired vascular repair as a possible cause of plaque vulnerability in diabetes. This notion is supported by observations of a reduced content of fibrous proteins and smooth muscle cell mitogens in carotid endarterectomy from diabetic patients along with findings of decreased circulating levels of endothelial progenitor cells. In the present study we used a diabetic mouse model to characterize how hyperglycemia affects arterial repair responses. We induced atherosclerotic plaque formation in ApoE-deficient (ApoE−/−) and heterozygous glucokinase knockout ApoE-deficient mice (ApoE−/− GK+/−) mice with a shear stress-modifying cast. There were no differences in cholesterol or triglyceride levels between the ApoE−/− and ApoE−/− GK+/− mice. Hyperglycemia did not affect the size of the formed atherosclerotic plaques, and no effects were seen on activation of cell proliferation, smooth muscle cell content or on the expression and localization of collagen, elastin and several other extracellular matrix proteins. The present study demonstrates that hyperglycemia per se has no significant effects on tissue repair processes in injured mouse carotid arteries, suggesting that other mechanisms are involved in diabetic plaque vulnerability.

Cyclic Mechanical Stretch Up-regulates Hepatoma-Derived Growth Factor Expression in Cultured Rat Aortic Smooth Muscle Cells.

Hepatoma-derived growth factor (HDGF) is a potent mitogen for vascular smooth muscle cells (SMCs) during embryogenesis and injury repair of vessel walls. Whether mechanical stimuli modulate HDGF expression remains unknown. The present study aimed at investigating whether cyclic mechanical stretch plays a regulatory role in HDGF expression and regenerative cytokine production in aortic SMCs. A SMC cell line was grown on a silicone-based elastomer chamber with extracellular matrix coatings (either type I collagen or fibronectin) and received cyclic and uniaxial mechanical stretches with 10% deformation at frequency 1 Hz. Morphological observation showed that fibronectin coating provided better cell adhesion and spreading and that consecutive 6 h of cyclic mechanical stretch remarkably induced reorientation and realignment of SMCs. Western blotting detection demonstrated that continuous mechanical stimuli elicited up-regulation of HDGF and proliferative cell nuclear antigen, a cell proliferative marker. Signal kinetic profiling study indicated that cyclic mechanical stretch induced signaling activity in RhoA/ROCK and PI3K/Akt cascades. Kinase inhibition study further showed that blockade of PI3K activity suppressed the stretch-induced tumor necrosis factor-α (TNF-α), whereas RhoA/ROCK inhibition significantly blunted the interleukin-6 (IL-6) production and HDGF overexpression. Moreover, siRNA-mediated HDGF gene silencing significantly suppressed constitutive expression of IL-6, but not TNF-α, in SMCs. These findings support the role of HDGF in maintaining vascular expression of IL-6, which has been regarded a crucial regenerative factor for acute vascular injury. In conclusion, cyclic mechanical stretch may maintain constitutive expression of HDGF in vascular walls and be regarded an important biophysical regulator in vascular regeneration.

Phosphatidylinositol 3-Kinase-DNA Methyltransferase 1-miR-1281-Histone Deacetylase 4 Regulatory Axis Mediates Platelet-Derived Growth Factor-Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells.

BackgroundPlatelet‐derived growth factor BB, a potent mitogen of pulmonary artery smooth muscle cells (PASMCs), has been implicated in pulmonary arterial remodeling, which is a key pathogenic feature of pulmonary arterial hypertension. Previous microRNA profiling in platelet‐derived growth factor BB–treated PASMCs found a significantly downregulated microRNA, miR‐1281, but it has not been associated with any cellular function, and we investigated the possibility.Methods and ResultsReal‐time quantitative reverse transcription–polymerase chain reaction assay proved that downregulation of miR‐1281 was a conserved phenomenon in human and rat PASMCs. Overexpression and inhibition of miR‐1281 in PASMCs promoted and suppressed, respectively, the cell proliferation and migration. Bioinformatic prediction and 3′‐untranslated region reporter assay identified histone deacetylase 4 to be a direct target of miR‐1281. Supporting this, proliferation and migration assay demonstrated the cellular function of histone deacetylase 4 is inversely correlated with that of miR‐1281. Mechanistically, it is found that platelet‐derived growth factor BB activates the phosphatidylinositol 3‐kinase pathway, which then induces the expression of DNA methyltransferase 1, leading to enhanced methylation of a flanking CpG island and repressed miR‐1281 expression. Finally, a reduced miR‐1281 level was consistently identified in hypoxic PASMCs in vitro, in pulmonary arteries of rats with monocrotaline‐induced pulmonary arterial hypertension, and in serum of patients with coronary heart disease–pulmonary arterial hypertension. These data suggest that there may be a diagnostic and therapeutic use for miR‐1281.ConclusionsHerein, we report a novel regulatory axis, phosphatidylinositol 3‐kinase–DNA methyltransferase 1–miR‐1281–histone deacetylase 4, integrating multiple epigenetic regulators that participate in platelet‐derived growth factor BB–stimulated PASMC proliferation and migration and pulmonary vascular remodeling.

Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling.

Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Smooth Muscle Insulin-Like Growth Factor-1 Mediates Hypoxia-Induced Pulmonary Hypertension in Neonatal Mice.

Insulin-like growth factor (IGF)-1 is a potent mitogen of vascular smooth muscle cells (SMCs), but its role in pulmonary vascular remodeling associated with pulmonary hypertension (PH) is not clear. In an earlier study, we implicated IGF-1 in the pathogenesis of hypoxia-induced PH in neonatal mice. In this study, we hypothesized that hypoxia-induced up-regulation of IGF-1 in vascular smooth muscle is directly responsible for pulmonary vascular remodeling and PH. We studied neonatal and adult mice with smooth muscle-specific deletion of IGF-1 and also used an inhibitor of IGF-1 receptor (IGF-1R), OSI-906, in neonatal mice. We found that, in neonatal mice, SMC-specific deletion of IGF-1 or IGF-1R inhibition with OSI-906 attenuated hypoxia-induced pulmonary vascular remodeling in small arteries, right ventricular hypertrophy, and right ventricular systolic pressure. Pulmonary arterial SMCs from IGF-1-deleted mice or after OSI-906 treatment exhibited reduced proliferative potential. However, in adult mice, smooth muscle-specific deletion of IGF-1 had no effect on hypoxia-induced PH. Our data suggest that vascular smooth muscle-derived IGF-1 plays a critical role in hypoxia-induced PH in neonatal mice but not in adult mice. We speculate that the IGF-1/IGF-1R axis is important in pathogenesis of PH in the developing lung and may be amenable to therapeutic manipulation in this age group.

Abstract 521: Janus Kinase 3 is a Novel Regulator for Smooth Muscle Proliferation and Vascularremodeling

Vascular remodeling due to smooth muscle cell (SMC) proliferation is a common process occurring in a number of vascular diseases such as atherosclerosis, aortic aneurysm, posttransplant vasculopathy, and restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completed understood. Our present study has identified Janus kinase 3 (JAK3), a member of the Janus kinase family, as a novel regulator for SMC proliferation. Platelet-derived growth factor (PDGF)-BB, a SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by shRNA inhibits SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Interestingly, JAK3 does not affect SMC contractile protein expression, suggesting that JAK3 mediate the proliferation, but not the phenotypic modulation of SMC. Mechanistically, JAK3 promotes SMC proliferation by regulating phosphorylation of Signal transducer and activator of transcription 3 and c-Jun N-terminal kinase. In vivo, by using a rat carotid balloon-injury model, we found that knockdown of JAK3 significantly attenuates injury-induced neointima formation. Importantly, JAK3 knockdown blocked the expression of proliferating cell nuclear antigen, suggesting that JAK3 is essential for SMC proliferation in vivo. Moreover, JAK3 knockdown prompts apoptosis in neointima SMC, indicating that JAK3 also stimulates SMC survival during neointima formation. Collectively, our data demonstrate that JAK3 mediates vascular remodeling by promoting both the SMC proliferation and survival.

Abstract 43: HBEGF cooperates with Cdkn2a and Pten loss to form malignant gliomas

Abstract Malignant gliomas are aggressive brain tumors that carry a grim prognosis to over 10,000 patients a year in the United States. The highest grade tumor, glioblastoma, has an average survival of one year with the current standard of care, which includes surgical resection, radiotherapy, and temozolomide chemotherapy. Heparin-binding EGF-like growth factor (HBEGF), first discovered in macrophages as a mitogen for vascular smooth muscle cells, is a ligand for epidermal growth factor receptor, the most commonly amplified receptor-tyrosine kinase in glioblastoma. About 30% of glioblastomas express HBEGF, and it is also commonly upregulated in tumors that express the common EGFR mutant, EGFRvIII. To date, HBEGF has not been evaluated for its ability to promote the development of malignant gliomas in vivo. Using a method for somatic cell gene transfer, we show that HBEGF cooperates with loss of tumor suppressors Cdkn2a and Pten to form malignant gliomas in vivo. These tumors share many characteristics found in human glioblastoma such as necrosis and nuclear atypia. We also demonstrate using high-throughput sequencing that these tumors share a common gene signature to the human glioblastoma samples from The Cancer Genome Atlas study. No tumors were seen with loss of Cdkn2a and Pten alone, which are the two most common tumor suppressors altered in glioblastoma. Our findings demonstrate that HBEGF can drive the development of high grade gliomas in the context of Cdkn2a and Pten loss and that these tumors closely resemble the human disease. Citation Format: Clifford Shin, Sheri Holmen. HBEGF cooperates with Cdkn2a and Pten loss to form malignant gliomas. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 43.

HMGB1-Driven Inflammation and Intimal Hyperplasia After Arterial Injury Involves Cell-Specific Actions Mediated by TLR4

Endoluminal vascular interventions such as angioplasty initiate a sterile inflammatory response resulting from local tissue damage. This response drives the development of intimal hyperplasia (IH) that, in turn, can lead to arterial occlusion. We hypothesized that the ubiquitous nuclear protein and damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), is one of the endogenous mediators that activates processes leading to IH after endoluminal injury to the arterial wall. The aim of this study is to investigate whether approaches that reduce the levels of HMGB1 or inhibit its activity suppresses IH after arterial injury. Here, we show that HMGB1 regulates IH in a mouse carotid wire injury model. Induced genetic deletion or neutralization of HMGB1 prevents IH, monocyte recruitment, and smooth muscle cell growth factor production after endoluminal carotid artery injury. A specific inhibitor of HMGB1 myeloid differentiation factor 2-toll-like receptor 4 (TLR4) interaction, P5779, also significantly inhibits IH. HMGB1 deletion is mimicked in this model by global deletion of TLR4 and partially replicated by myeloid-specific deletion of TLR4 but not TLR2 or receptor for advanced glycation endproducts deletion. The specific HMGB1 isoform known to activate TLR4 signaling (disulfide HMGB1) stimulates smooth muscle cell to migrate and produce monocyte chemotactic protein 1/CCL2) via TLR4. Macrophages produce smooth muscle cell mitogens in response to disulfide HMGB1 also in a TLR4/myeloid differentiation primary response gene (88)/Trif-dependent manner. These findings place HMGB1 and its receptor, TLR4 as critical regulators of the events that drive the inflammation leading to IH after endoluminal arterial injury and identify this pathway as a possible therapeutic target to limit IH to attenuate damage-associated molecular pattern molecule-mediated vascular inflammatory responses.

PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (VSMCs). However, the direct effects of PDGF receptor β (PDGFRβ) activation on VSMCs have not been studied in the context of atherosclerosis. Here, we present a new mouse model of atherosclerosis with an activating mutation in PDGFRβ. Increased PDGFRβ signaling induces chemokine secretion and leads to leukocyte accumulation in the adventitia and media of the aorta. Furthermore, PDGFRβD849V amplifies and accelerates atherosclerosis in hypercholesterolemic ApoE−/− or Ldlr−/− mice. Intriguingly, increased PDGFRβ signaling promotes advanced plaque formation at novel sites in the thoracic aorta and coronary arteries. However, deletion of the PDGFRβ-activated transcription factor STAT1 in VSMCs alleviates inflammation of the arterial wall and reduces plaque burden. These results demonstrate that PDGFRβ pathway activation has a profound effect on vascular disease and support the conclusion that inflammation in the outer arterial layers is a driving process for atherosclerosis.

Tissue transglutaminase promotes serotonin-induced AKT signaling and mitogenesis in pulmonary vascular smooth muscle cells

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50μM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling.

Effects of serotonin on expression of the LDL receptor family member LR11 and 7-ketocholesterol-induced apoptosis in human vascular smooth muscle cells

Serotonin (5-HT) is a known mitogen for vascular smooth muscle cells (VSMCs). The dedifferentiation and proliferation/apoptosis of VSMCs in the arterial intima represent one of the atherosclerotic changes. LR11, a member of low-density lipoprotein receptor family, may contribute to the proliferation of VSMCs in neointimal hyperplasia. We conducted an in vitro study to investigate whether 5-HT is involved in LR11 expression in human VSMCs and apoptosis of VSMCs induced by 7-ketocholesterol (7KCHO), an oxysterol that destabilizes plaque. 5-HT enhanced the proliferation of VSMCs, and this effect was abolished by sarpogrelate, a selective 5-HT2A receptor antagonist. Sarpogrelate also inhibited the 5-HT-enhanced LR11 mRNA expression in VSMCs. Furthermore, 5-HT suppressed the 7KCHO-induced apoptosis of VSMCs via caspase-3/7-dependent pathway.These findings provide new insights on the changes in the differentiation stage of VSMCs mediated by 5-HT.