Hpasmc

Manufactured by Thermo Fisher Scientific

Sourced in United States, Switzerland

HPASMCs are human pulmonary artery smooth muscle cells. They are primary cells derived from human pulmonary arteries and are designed for in vitro research applications.

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12 protocols using hpasmc

Hypoxia regulation of hPASMCs via SIK1

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Primary human pulmonary arterial smooth muscle cells (hPASMCs, c-12521) were purchased from American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM, 1101, ScienCell) supplemented with 2.5% fetal bovine serum (FBS), 1% penicillin/streptomycin (PS) solution and 1% smooth muscle cell growth supplement (SMCGS). All cells were grown at 37 °C in humidified air with 5% CO₂. Cells at passages 3–9 were used for the experiments.
For hypoxia (3% O₂) experiments, hPASMCs were cultured in serum-free DMEM for 12 h before treatments and then placed in a Heracell Vios 150i CO₂ incubator (Thermo Fisher Scientific) for 24 h. For the normoxia control, hPASMCs were cultured in normal incubators with 21% O₂ for 24 h.
Transfections were performed using Lipofectamine® 3000 Transfection Reagent (L3000075, Invitrogen, CA, USA) according to the manufacturers’ instructions. Short-interfering RNAs targeting SIK1 (SIK1 siRNA) (sense: 5′-CCACUUUGCUGCCAUUUAUTT-3′, antisense: 5′-AUAAAUGGCAGCAAAGUGGTT-3′) and a relative scrambled siRNA were designed and synthesized by GenePharma (China). Human YAP siRNA (sc-38637) was purchased from Santa Cruz Biotechnology. SIK1 adenovirus (Ad SIK1) and null adenovirus (Ad null) were purchased from GeneChem (China). Ad SIK1 was used to overexpress SIK1.

Pu J., Wang F., Ye P., Jiang X., Zhou W., Gu Y, & Chen S. (2022). Salt-inducible kinase 1 deficiency promotes vascular remodeling in pulmonary arterial hypertension via enhancement of yes-associated protein-mediated proliferation. Heliyon, 8(10), e11016.

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HPASMC Cigarette Smoke Epigenetics

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HPASMCs were purchased from American Type Culture Collection (MD, USA) and grown in Dulbecco’s Modified Eagle’s Medium-F12 containing 10% fetal bovine serum. Cigarette smoke extract (CSE) obtained from Research Cigarettes (Code 3R4F, University of Kentucky, USA) was acquired as described elsewhere [35 (link)]. DNMT1 small interfering RNA (siRNA; 50 nM) was transfected into HPASMCs using Lipofectamine 2000 (Invitrogen, USA) for silencing DNMT1. DNMT1 siRNA target sequences were as follows: the first 5′-GCACCUCAUUUGCCGAAUATT-3′; the second 5′- GGGACUGUGUCUCUGUUAUTT-3′. DNMT1 overexpression pcDNA3.1 plasmid vector was also transfected into HPASMCs using Lipofectamine 2000 (Vigene Biosciences, China). The RASEF overexpression adenovirus vector (Ad.RASEF; Vigene Biosciences, China) was also transfected into HPASMCs (MOI 250).

Li Q., Wu J., Xu Y., Liu L, & Xie J. (2019). Role of RASEF hypermethylation in cigarette smoke-induced pulmonary arterial smooth muscle remodeling. Respiratory Research, 20, 52.

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SIRT1 Overexpression and Knockdown in HPASMCs

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HPASMCs (Invitrogen) were cultured in M231 medium (Invitrogen) with smooth muscle growth supplement. The replication-defective adenoviral vectors expressing SIRT1 (Ad-SIRT1) or control green fluorescent protein (Ad-GFP) and adenovirus-mediated knockdown of SIRT1 (Ad-SIRT1 RNAi) or control vector (Ad-U6) were generated as described previously [26 (link), 32 (link)]. HPASMCs were infected for 2 hours with the above adenovirus using a multiplicity of infection (MOI) of 100, washed, and incubated in serum-free medium without virus for at least 24 hours before drugs challenge.

Zhou S., Li M.T., Jia Y.Y., Liu J.J., Wang Q., Tian Z., Liu Y.T., Chen H.Z., Liu D.P, & Zeng X.F. (2015). Regulation of Cell Cycle Regulators by SIRT1 Contributes to Resveratrol-Mediated Prevention of Pulmonary Arterial Hypertension. BioMed Research International, 2015, 762349.

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Modulating GPS2 in Human Pulmonary Artery Smooth Muscle Cells

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Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from Proximity Life Sciences (China). The cells were cultured in DMEM solution containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and placed in a constant temperature incubator at 37 °C with 5% CO2. HPASMCs were infected with Ad.GPS2 (adenovirus for GPS2 overexpression) and NC.Ad (the control adenovirus) (MOI = 200); and we also transfected HPASMCs with GPS2 siRNA (siGPS2; 50 nM) using Lipofectamine 2000 (Invitrogen, USA) for silencing GPS2. And SiGPS2 target sequences were as follows: the first 5′-CAGCCAGCTTATAGTCCTA − 3′, the second 5′-GGAGAAGCTTTTGGCTCTA − 3′.

Hu T., Mu C., Li Y., Hao W., Yu X., Wang Y., Han W, & Li Q. (2024). GPS2 ameliorates cigarette smoking-induced pulmonary vascular remodeling by modulating the ras-Raf-ERK axis. Respiratory Research, 25, 210.

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Culturing and Hypoxic Stimulation of hPASMCs

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Human pulmonary artery smooth muscle cells (hPASMCs; Cascade Biologics Inc., Portland, OR) were cultured in SmGM-2 BulletKit media (Lonza, Basel, Switzerland) containing 5% (volume/volume) heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA), 0.5 ng/ml human recombinant epidermal growth factor, 2 ng/ml human recombinant fibroblast growth factor, 5 μg/ml insulin, and 50 μg/ml gentamicin. HEK293 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere containing 5% CO₂. Cells at passages 6–8 were used for experiments. For induction of hypoxia, cells were transferred in a special hypoxia incubator (Thermo Scientific, model 3130, Rockford, IL) with 3% O₂, 5% CO₂, and balanced nitrogen. The O₂ concentration inside the chamber was detected continuously by using an oxygen monitor (Hudson Ventronics Division, CA) to ensure that the O₂ concentration was 3%.

Lu Z., Li S., Zhao S, & Fa X. (2016). Upregulated miR-17 Regulates Hypoxia-Mediated Human Pulmonary Artery Smooth Muscle Cell Proliferation and Apoptosis by Targeting Mitofusin 2. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 3301-3308.

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Culturing Human Pulmonary Artery Smooth Muscle Cells

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Human pulmonary artery smooth muscle cells (hPASMCs), purchased from Cascade Biologics Inc (Portland, Oregon, USA), were cultured with SmGM‐2 BulletKit media (Lonza Japan, Tokyo, Japan) supplemented with 5% foetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified incubator with 5% CO₂ at 37˚C. For hypoxia environment, hPASMCs were exposed to 1% O₂ for indicated times.

Song R., Lei S., Yang S, & Wu S. (2021). LncRNA PAXIP1‐AS1 fosters the pathogenesis of pulmonary arterial hypertension via ETS1/WIPF1/RhoA axis. Journal of Cellular and Molecular Medicine, 25(15), 7321-7334.

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Modulating hPASMCs with DPP4 siRNA

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hPASMCs were purchased from PromoCell (Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (PromoCell) supplemented with 10% fetal bovine serum. hPASMCs were incubated at 37 °C in a 5% CO₂ incubator and used at passages 4–6 for all experiments. For small interfering RNA (siRNA) transfection, DPP4 siRNA (Cat# 4392421, siRNA ID: s4255) and non-specific control siRNA (Cat# 4390843, Silencer™ Select Negative Control No. 1 siRNA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Using the Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific), hPASMCs were transfected with siRNA for 48 h according to the manufacturer’s protocol. After the siRNA treatment, the cells were stimulated with recombinant human transforming growth factor-b1 (TGFβ) (PEPROTECH. Cranbury, NJ, USA) at a concentration of 10 ng/mL as previously reported [45 (link)] or with PBS at the same concentration for 24 h.

Okaya T., Kawasaki T., Sato S., Koyanagi Y., Tatsumi K., Hatano R., Ohnuma K., Morimoto C., Kasuya Y., Hasegawa Y., Ohara O, & Suzuki T. (2024). Functional Roles of CD26/DPP4 in Bleomycin-Induced Pulmonary Hypertension Associated with Interstitial Lung Disease. International Journal of Molecular Sciences, 25(2), 748.

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Evaluating Endothelial and Smooth Muscle Cell Responses

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Human pulmonary microvascular endothelial cells (HPMECs; cat. no., 3000) were purchased from ScienCell Research Laboratories, Inc. (San Diego, CA, USA), while human pulmonary artery smooth muscle cells (HPASMCs) were obtained from iCell Bioscience, Inc. (Shanghai, China). All cells were cultured and subcultured according to the manufacturer’s protocol. HPMECs were incubated in Endothelial Cell medium (ScienCell Research Laboratories, Inc.) and HPASMCs were maintained in Dulbecco’s Modified Eagle’s Medium and high glucose medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C under an atmosphere of 5% CO₂ and 95% air. Cells were used in the third passage at a concentration of 5×10⁵ cells per well in a 12 well-plate, or 1×10⁶ cells per well in a 6-well plate. Cells were pre-stimulated with fasudil (10 µM) for 2 h, followed by incubation with ET-1 (10 nM; cat no. 1160; Tocris Bioscience, Bristol, UK) for a further 6 h for the migration assay. PBS-only treated cells were used as the control. HPMECs were used to confirmed the nitric oxide (NO) production and migration, while HPASMCs were used to detect the effect of fasudil on the proliferation of SMC.

Zhuang R., Wu J., Lin F., Han L., Liang X., Meng Q., Jiang Y., Wang Z., Yue A., Gu Y., Fan H., Zhou X, & Liu Z. (2018). Fasudil preserves lung endothelial function and reduces pulmonary vascular remodeling in a rat model of end-stage pulmonary hypertension with left heart disease. International Journal of Molecular Medicine, 42(3), 1341-1352.

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Investigating Pulmonary Artery Smooth Muscle Cells

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Human Pulmonary Artery Smooth Muscle Cells (hPASMCs) and smooth muscle cell medium (SMCM) were purchased from Sciencell (CA, USA; catalog #3110 and #1101). hPASMCs were placed in a CO₂ incubator (5%, Thermo, USA) with a humidified atmosphere containing 5% CO₂-95% air at 37°C in complete SMCM containing 5% fetal bovine serum (FBS, Gibco; Grand Island, NY, USA).
For CSE treatment, after cells had grown to 50–60% confluence, the culture media was replaced with SMCM basal culture media supplemented with 0.3% FBS for 12–24 h, and then exposed to 0, 0.125, 0.25, 0.5, 1, 2, 4% of CSE stimuli for 24, 36, 48 h, respectively.

He W., Liu C., Liao J., Liu F., Lei H., Wei D., Ruan H., Kunwar B., Lu W., Wang J, & Wang T. (2022). TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients. Frontiers in Medicine, 8, 774623.

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Transient Transfection of Human Pulmonary Artery Smooth Muscle Cells

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Human pulmonary artery smooth muscle cell (HPASMc) was purchased from Sciencell Company (San Diego, CA, USA). The smooth muscle cell medium (SMCM) (Gibco/ThermoFisher, Waltham, MI, USA) for HPASMc culturing was at 37 °C in a humidified atmosphere with 95% air and 5% CO₂.
In transient transfection test, HPASM cells were pre-cultured in Dulbecco’s modified eagle medium (DMEM) (Gibco/ThermoFisher) without serum. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were adapted to transfect 3.2 μg of each constructed vector into cells in 6-well plate. We changed the complete medium for further culture after six hours.

Xu M., Xu M., Han L., Yuan C., Mei Y., Zhang H., Chen S., Sun K, & Zhu B. (2017). Role for Functional SOD2 Polymorphism in Pulmonary Arterial Hypertension in a Chinese Population. International Journal of Environmental Research and Public Health, 14(3), 266.

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