Procyanidin B2 Reduces Vascular Calcification through Inactivation of ERK1/2-RUNX2 Pathway

Vascular calcification is strongly associated with atherosclerotic plaque burden and plaque instability. The activation of extracellular signal-regulated kinase 1/2 (ERK1/2) increases runt related transcription factor 2 (RUNX2) expression to promote vascular calcification. Procyanidin B2 (PB2), a potent antioxidant, can inhibit ERK1/2 activation in human aortic smooth muscle cells (HASMCs). However, the effects and involved mechanisms of PB2 on atherosclerotic calcification remain unknown. In current study, we fed apoE-deficient (apoE−/−) mice a high-fat diet (HFD) while treating the animals with PB2 for 18 weeks. At the end of the study, we collected blood and aorta samples to determine atherosclerosis and vascular calcification. We found PB2 treatment decreased lesions in en face aorta, thoracic, and abdominal aortas by 21.4, 24.6, and 33.5%, respectively, and reduced sinus lesions in the aortic root by 17.1%. PB2 also increased α-smooth muscle actin expression and collagen content in lesion areas. In the aortic root, PB2 reduced atherosclerotic calcification areas by 75.8%. In vitro, PB2 inhibited inorganic phosphate-induced osteogenesis in HASMCs and aortic rings. Mechanistically, the expression of bone morphogenetic protein 2 and RUNX2 were markedly downregulated by PB2 treatment. Additionally, PB2 inhibited ERK1/2 phosphorylation in the aortic root plaques of apoE−/− mice and calcified HASMCs. Reciprocally, the activation of ERK1/2 phosphorylation by C2-MEK1-mut or epidermal growth factor can partially restore the PB2-inhibited RUNX2 expression or HASMC calcification. In conclusion, our study demonstrates that PB2 inhibits vascular calcification through the inactivation of the ERK1/2-RUNX2 pathway. Our study also suggests that PB2 can be a potential option for vascular calcification treatment.

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Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2

Bioscience Reports ◽

10.1042/bsr20190599 ◽

2019 ◽

Vol 39 (10) ◽

Author(s):

Ashish Patidar ◽

Dhruv K. Singh ◽

Shori Thakur ◽

Ken Farrington ◽

Anwar R. Baydoun

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Density Lipoprotein ◽

Hdl Cholesterol ◽

Muscle Cells ◽

Runx2 Expression ◽

Cholesterol Ratio ◽

Ckd Stage ◽

Related Transcription Factor

Abstract Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and β-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

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Abstract 348: Dietary Quercetin Prevents Warfarin-Induced Vascular Calcification in Rats

Circulation Research ◽

10.1161/res.111.suppl_1.a348 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Maria Nurminskaya ◽

Derek Banyard ◽

Dmitry Nurminsky ◽

Mikhail Konoplyannikov ◽

Kelly Beazley

Keyword(s):

Smooth Muscle ◽

Vascular Calcification ◽

Vitamin K ◽

Smooth Muscle Actin ◽

Male Rats ◽

Arterial Calcification ◽

Type I ◽

Blood Pressure Elevation

Medial arterial calcification contributes to the development of isolated systolic hypertension, the most frequent type of blood pressure elevation in the elderly; complicates a variety of prevalent disorders such as diabetes and renal disease; and can develop in response to warfarin - the staple in anticoagulant therapy to millions of people. Our in vitro studies have shown that the bioflavonoid quercetin effectively prevents warfarin-induced vascular calcification in VSMCs and identified b-catenin signaling as a specific target of this effect. Here, we have tested in vivo the association of b-catenin signaling with warfarin-induced calcification and the efficacy of quercetin in preventing warfarin-induced aortic calcium accrual. Vitamin K-warfarin treatment was used for 4 weeks to induce arterial calcification in male rats. Molecular analysis demonstrates activation of b-catenin signaling in the calcified areas and osteoblast-like transformation in vascular cells. Dietary quercetin (10 mg/kg body weight) (Quercegen Pharma, Newton, MA) efficiently prevents warfarin-induced calcification (3.04+/-0.45 ug Ca in vitamin K-warfarin animals vs 0.77+/-0.08 ug Ca in quercetin supplementation per mg dry arterial tissue (n=5; p<0.05). In parallel, quercetin inhibits b-catenin signaling and phenotypic transformation in vascular smooth muscle analyzed by expression of transcription factor Runx2, collagen type I and osteocalcin. Further, quercetin diet enhances expression of VSMC markers smooth muscle actin, myosin heavy chain, sm22a and calponin, suggesting a stabilized smooth muscle phenotype of these cells. No toxic or systemic effects of quercetin treatment were observed, identifying this bioflavonoid as a putative therapeutic for vascular calcification.

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Estrogen inhibits vascular calcification in rats via hypoxia-induced factor-1α signaling

Vascular ◽

10.1177/1708538120904297 ◽

2020 ◽

Vol 28 (4) ◽

pp. 465-474

Author(s):

Xinhua Wu ◽

Qiuyan Zhao ◽

Zhangrong Chen ◽

Yong-Jian Geng ◽

Wanting Zhang ◽

...

Keyword(s):

Smooth Muscle ◽

Coronary Artery ◽

Bone Morphogenetic Protein ◽

Vascular Calcification ◽

Coronary Artery Calcification ◽

Bone Morphogenetic Protein 2 ◽

Ovariectomized Rats ◽

Sprague Dawley ◽

Morphogenetic Protein

Objective Calcification serves as a surrogate for atherosclerosis-associated vascular diseases, and coronary artery calcification is mediated by multiple pathogenic factors. Estrogen is a known factor that protects the arterial wall against atherosclerosis, but its role in the coronary artery calcification development remains largely unclear. This study tested the hypothesis that estrogen inhibits coronary artery calcification via the hypoxia-induced factor-1α pathway. Methods Eight-week-old healthy female Sprague–Dawley rats were castrated, and vitamin D3 was administered orally to establish. Hypoxia-induced factor-1 inhibitor was administered to test its effect on vascular calcification and expression of bone morphogenetic protein 2 and runt-related transcription factor-2. Vascular smooth muscle cell calcification was induced with CaCl2 in rat aortic smooth muscle cells in the presence or absence of E2(17β-estradiol) and bone morphogenetic protein 2 siRNA intervention. Results The estrogen levels in ovariectomized rats were significantly decreased, as determined by ELISA. Expression of hypoxia-induced factor-1α mRNA and protein was significantly increased in vascular cells with calcification as compared to those without calcification ( p < 0.01). E2 treatment decreased the calcium concentration in vascular cell calcification and cell calcium nodules in vitro ( p < 0.05). E2 also lowered the levels of hypoxia-induced factor-1α mRNA and protein ( p < 0.01). Oral administration of the hypoxia-induced factor-1α inhibitor dimethyloxetane in castrated rats alleviated vascular calcification and expression of osteogenesis-related transcription factors, bone morphogenetic protein 2 and RUNX2 ( p < 0.01). Finally, bone morphogenetic protein 2 siRNA treatment decreased the levels of p-Smad1/5/8 in A7r5 calcification cells ( p < 0.01). Conclusion Estrogen deficiency enhances vascular calcification. Treatment with estrogen reduces the expression of hypoxia-induced factor-1α as well as vascular calcification in rats. The estrogen effects occur in a fashion dependent on hypoxia-induced factor-1α regulation of bone morphogenetic protein-2 and downstream Smad1/5/8.

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Large intraperitoneal lipoleiomyoma in a pre-menopausal woman: a case report

World Journal of Surgical Oncology ◽

10.1186/s12957-021-02256-9 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Sara L. Schaefer ◽

Amy L. Strong ◽

Sheena Bahroloomi ◽

Jichang Han ◽

Michella K. Whisman ◽

...

Keyword(s):

Differential Diagnosis ◽

Smooth Muscle ◽

Case Report ◽

Smooth Muscle Actin ◽

Abdominal Mass ◽

Histopathological Analysis ◽

Menopausal Woman ◽

En Bloc ◽

Cross Sectional

Abstract Background Lipoleiomyoma is a rare, benign variant of the commonplace uterine leiomyoma. Unlike leiomyoma, these tumors are composed of smooth muscle cells admixed with mature adipose tissue. While rare, they are most frequently identified in the uterus, but even more infrequently have been described in extrauterine locations. Case presentation We describe a case report of a 45-year-old woman with a history of in vitro fertilization pregnancy presenting 6 years later with abdominal distention and weight loss found to have a 30-cm intra-abdominal lipoleiomyoma. While cross-sectional imaging can narrow the differential diagnosis, histopathological analysis with stains positive for smooth muscle actin, desmin, and estrogen receptor, but negative for HMB-45 confirms the diagnosis of lipoleiomyoma. The large encapsulated tumor was resected en bloc. The patients post-operative course was uneventful and her symptoms resolved. Conclusions Lipoleiomyoma should be considered on the differential diagnosis in a woman with a large intra-abdominal mass. While considered benign, resection should be considered if the mass is symptomatic, and the diagnosis is unclear or there is a concern for malignancy.

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Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.418 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Martin Liu ◽

Angelos Karagiannis ◽

Matthew Sis ◽

Srivatsan Kidambi ◽

Yiannis Chatzizisis

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Type I Collagen ◽

Smooth Muscle Actin ◽

Muscle Cells ◽

Type I ◽

Collagen Gels ◽

Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

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MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab086.0021 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Jana Holmar ◽

Heidi Noels ◽

Joachim Jankowski ◽

Setareh Orth-Alampour

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Calcification ◽

Vascular Smooth Muscle Cells ◽

Incubation Time ◽

Fetal Calf Serum ◽

Ex Vivo ◽

Calf Serum

Abstract Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

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The Functional Role of Zinc Finger E Box-Binding Homeobox 2 (Zeb2) in Promoting Cardiac Fibroblast Activation

International Journal of Molecular Sciences ◽

10.3390/ijms19103207 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3207 ◽

Cited By ~ 3

Author(s):

Fahmida Jahan ◽

Natalie Landry ◽

Sunil Rattan ◽

Ian Dixon ◽

Jeffrey Wigle

Keyword(s):

Smooth Muscle ◽

Zinc Finger ◽

Cardiac Fibrosis ◽

Smooth Muscle Actin ◽

Critical Role ◽

Cardiac Fibroblast ◽

In Vivo Studies ◽

Fibroblast Activation ◽

E Box

Following cardiac injury, fibroblasts are activated and are termed as myofibroblasts, and these cells are key players in extracellular matrix (ECM) remodeling and fibrosis, itself a primary contributor to heart failure. Nutraceuticals have been shown to blunt cardiac fibrosis in both in-vitro and in-vivo studies. However, nutraceuticals have had conflicting results in clinical trials, and there are no effective therapies currently available to specifically target cardiac fibrosis. We have previously shown that expression of the zinc finger E box-binding homeobox 2 (Zeb2) transcription factor increases as fibroblasts are activated. We now show that Zeb2 plays a critical role in fibroblast activation. Zeb2 overexpression in primary rat cardiac fibroblasts is associated with significantly increased expression of embryonic smooth muscle myosin heavy chain (SMemb), ED-A fibronectin and α-smooth muscle actin (α-SMA). We found that Zeb2 was highly expressed in activated myofibroblast nuclei but not in the nuclei of inactive fibroblasts. Moreover, ectopic Zeb2 expression in myofibroblasts resulted in a significantly less migratory phenotype with elevated contractility, which are characteristics of mature myofibroblasts. Knockdown of Zeb2 with siRNA in primary myofibroblasts did not alter the expression of myofibroblast markers, which may indicate that Zeb2 is functionally redundant with other profibrotic transcription factors. These findings add to our understanding of the contribution of Zeb2 to the mechanisms controlling cardiac fibroblast activation.

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A translational cellular model for the study of peritubular cells of the testis

Reproduction ◽

10.1530/rep-20-0100 ◽

2020 ◽

Vol 160 (2) ◽

pp. 259-268 ◽

Cited By ~ 2

Author(s):

Nina Schmid ◽

Annika Missel ◽

Stoyan Petkov ◽

Jan B Stöckl ◽

Florian Flenkenthaler ◽

...

Keyword(s):

Smooth Muscle ◽

Replicative Senescence ◽

Smooth Muscle Actin ◽

Proteome Analysis ◽

Inflammatory Factors ◽

Seminiferous Tubules ◽

Cellular Model ◽

Mechanistic Studies ◽

Peritubular Cells

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs. However, like their human counterparts these cells age in vitro and replicative senescence limits in-depth functional or mechanistic studies. Therefore, a stable cellular model was established. MKTPCs of a young adult animal were immortalized by piggyBac transposition of human telomerase (hTERT), that is, without the expression of viral oncogenes. Immortalized MKTPCs (iMKTPCs) grew without discernable changes for more than 50 passages. An initial characterization revealed typical genes expressed by peritubular cells (androgen receptor (AR), smooth-muscle actin (ACTA2), calponin (CNN1)). A proteome analysis of the primary MKTPCs and the derived immortalized cell line confirmed that the cells almost completely retained their phenotype. To test whether they respond in a similar way as HTPCs, iMKTPCs were challenged with forskolin (FSK) and ATP. As HTPCs, they showed increased expression level of the StAR protein (StAR) after FSK stimulation, indicating steroidogenic capacity. ATP increased the expression of pro-inflammatory factors (e.g. IL1B; CCL7), as it is the case in HTPCs. Finally, we confirmed that iMKTPCs can efficiently be transfected. Therefore, they represent a highly relevant translational model, which allows mechanistic studies for further exploration of the roles of testicular peritubular cells.

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Abstract 064: The Lipoxigenase Inhibitor Nordihydroguaiaretic Acid Prevents the Development of Hypoxia-Induced Pulmonary Hypertension in Mice

Circulation Research ◽

10.1161/res.113.suppl_1.a064 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Andrea Iorga ◽

Gabriel Wong ◽

Denise Mai ◽

Jingyuan Li ◽

Salil Sharma ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Smooth Muscle ◽

Smooth Muscle Actin ◽

Nordihydroguaiaretic Acid ◽

Treated Group ◽

Oxidation Products ◽

Lipoxygenase Inhibitor ◽

Human Pulmonary Artery

Pulmonary hypertension (PH) is a chronic lung disease characterized by progressively elevated pulmonary arterial pressures and severe pulmonary vascular remodeling resulting from interactions between oxidized lipoprotein deposition and increased endothelial proliferation. Previously we have shown increased plasma levels of biological oxidation products such as hydroxyoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acids (HETEs) in the rat monocrotaline model of PH. Here we investigated the role of HETEs and HODEs in the development of PH and whether their inhibition with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) attenuates the progression of PH. Mice were placed in a hypoxic chamber with O2 concentrations of ≤10% for 21 days and either left untreated to develop PH (n=7) or treated with NDGA daily (10mg/kg/day, i.p., n=4) from day 1. Direct RV catheterization was terminally performed to record RV pressure (RVP). Pulmonary arteriolar thickening and oxidized lipid deposition were assessed by staining lung sections with Masson’s Trichrome or with α-smooth muscle actin and E-06 (marker for oxidized low-density lipoproteins). In vitro, human pulmonary artery smooth muscle cell (hPASMC) proliferation was assessed by MTT assays in the absence or presence of 12-HETE (100ng/ml), 9-HODE (1µg/ml) and 13-HODE (1µg/ml) alone or together with NDGA (10, 25 and 50µM). In-vitro, HETE/HODE treatment increased hPASMC proliferation ~ 2-fold when compared to untreated cells and NDGA significantly inhibited the proliferative effects of all three oxidized lipids. In-vivo, NDGA treatment prevented the development of PH. RVP was lower in the NDGA-treated group vs. the PH group (24.01±1.39mmHg vs. 36.91±5.74mmHg, p<0.05) and was comparable to control normoxic mice (20.93±2.52mmHg). RV hypertrophy index was significantly elevated in the PH mice versus control mice (0.38±0.03 vs. 0.28±0.02 (p<0.001), while NDGA treatment completely prevented the development of RV hypertrophy (0.28±0.04). Lung sections demonstrated arteriolar thickening and E-06 positive deposits in the PH group, which was prevented by NDGA therapy. We conclude that oxidized fatty acid deposition and accumulation might play a role in the development of PH.

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Abstract 443: Constitutive Bone Morphogenetic Protein 2-dependent Osteogenic Signaling in M1 Macrophages

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.443 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Prabhatchandra Dube

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Er Stress ◽

Vascular Calcification ◽

Transient Receptor Potential ◽

Muscle Cells ◽

Bone Morphogenetic Protein 2 ◽

Osteogenic Potential ◽

M1 Macrophages ◽

Osteogenic Signaling

Atherosclerosis is the leading cause of carotid and coronary artery disease. Calcification often complicates atherosclerotic plaques and contributes to plaque instability.Mechanisms calcification are somewhat reminiscent of bone formation, involving interplay between endothelium, smooth muscle cells and plaque macrophages. Macrophages contribute to vascular calcification through signals that stimulate the osteogenic program in smooth muscle cells and/or by becoming osteoclast-like cells. Yet, the specific roles of polarized M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages, predominant in plaques, in lesion calcification remain poorly understood. Recent studies from our laboratory show that the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) is a key component of mechanisms linked to ER stress signaling in M1, but not M2 macrophages. Because ER stress has a recognized effect in inducing vascular calcification, we speculated that TRPC3, by virtue of its roles in M1 macrophages, might have an effect on the osteogenic potential of these cells. To address this question we utilized bone marrow derived macrophages from mice with macrophage-specific loss of TRPC3 function (MacTRPC3KO) or from their littermate controls (TRPC3 lox/lox ) and polarized in vitro to the M1 and M2 phenotypes. Osteogenic proteins and factors along with signaling mechanisms from the two groups were examined under basal and ER stress conditions. The results showed reduced expression of BMP-2 and Runx-2 at mRNA level in MacTRPC3KO M1 macrophages but not at the protein level between the groups. We also examined the phosphorylation status of SMAD1/5 in M1 macrophages, which is an early indicator of signaling downstream the BMP-2 receptor. Although no differences were observed between groups, SMAD1/5 was significantly phosphorylated, even under basal conditions. BMP-2 levels in supernatants from cultures of M1 macrophages was also significantly elevated regardless of the treatment condition. Notably, phosphorylation levels of SMAD1/5 were markedly reduced when cells were exposed to the BMP-2 receptor blocker dorsomorphin. These results indicate that BMP-2 is involved in activating osteogenic signaling in M1 macrophages.

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