Journal: Journal of Cardiovascular Pharmacology

Article Title: circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

doi: 10.1097/FJC.0000000000001396

Figure Lengend Snippet: circNFXL1 reversed the hypoxia-induced alterations in membrane potential and Kv currents in human pulmonary arterial smooth muscle cells (hPASMCs). A, circNFXL1 partially reversed the hypoxia-induced depolarization of resting membrane potential ( E m) in hPASMCs. The representative traces (B) and current-voltage relationships (IV) (C) of whole-cell Kv currents were recorded from hPASMCs in normoxia, hypoxia, and hypoxia+circNFXL1 conditions. D, The average amplitude of Kv currents recorded at +100 mV was compared between the 3 groups. * P < 0.05, ** P < 0.01, *** P < 0.001 (n = 10–12 cells). Mean ± SEM. Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post-hoc. ANOVA, analysis of variance.

Article Snippet: Detection of intracellular Ca 2+ was conducted by imaging the fluorescence intensity of fluo-loaded hPASMCs (OLYMPUS, BX51).

Techniques:

Journal: Journal of Cardiovascular Pharmacology

Article Title: circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

doi: 10.1097/FJC.0000000000001396

Figure Lengend Snippet: circNFXL1-regulated Kv2.1 expression via miR-29b-2 under hypoxic conditions. A, qPCR and (B) WB analysis of Kv2.1 in hPASMCs transfected with circNFXL1 expression vector or circNFXL1+miR-29b-2 mix and cultured under hypoxic conditions for 24–48 hours (hypoxia/circNFXL1, hypoxia/circNFXL1 + miR29b). hPASMCs transfected with pLC5-ciR and cultured under normal/hypoxic conditions were used as controls (normoxia/hypoxia). ** P < 0.01(n = 5 individual experiments). Mean ± SEM. Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post-hoc. ANOVA, analysis of variance.

Article Snippet: Detection of intracellular Ca 2+ was conducted by imaging the fluorescence intensity of fluo-loaded hPASMCs (OLYMPUS, BX51).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture

Journal: Journal of Cardiovascular Pharmacology

Article Title: circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

doi: 10.1097/FJC.0000000000001396

Figure Lengend Snippet: circNFXL1-regulated intracellular calcium via sponging miR-29b-2 under hypoxic conditions. Intracellular calcium level was measured using the Fluo-3-AM staining. The hPASMCs transfected with circNFXL1 expression vector or circNFXL1+miR-29b-2 mix and cultured under hypoxic conditions for 24–48 hours (hypoxia/circNFXL1, hypoxia/circNFXL1+miR29b). hPASMCs transfected with pLC5-ciR and cultured under normal/hypoxic conditions were used as controls (normoxia/hypoxia). After transfections, the cultured hPASMCs were labeled by Fura-3-AM. Detection of intracellular Ca 2+ was carried out by imaging the fluorescence intensity of fluo-loaded hPASMCs. * P < 0.05, ** P < 0.01 (n = 8–10 cells). Mean ± SEM. Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post-hoc. ANOVA, analysis of variance.

Article Snippet: Detection of intracellular Ca 2+ was conducted by imaging the fluorescence intensity of fluo-loaded hPASMCs (OLYMPUS, BX51).

Techniques: Staining, Transfection, Expressing, Plasmid Preparation, Cell Culture, Labeling, Imaging, Fluorescence

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification and validation of CCL5 as a key gene in HIV infection and pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1417701

Figure Lengend Snippet: Expression of CCL5 in hPAECs and hPASMCs. ( A ) Expression level of CCL5 between control and hypoxia groups in hPAECs. ( B ) Expression level of CCL5 between control and hypoxia groups in hPASMCs. hPAECs, human pulmonary artery endothelial cells; hPASMCs, human pulmonary artery smooth muscle cells. n = 3, * P < 0.05, ** P < 0.01.

Article Snippet: hPAECs were purchased from ScienCell (Shanghai, China, Cat. No. 3100). hPASMCs were obtained from Procell (Wuhan, China, Cat. No. CP-H243).

Techniques: Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: Sildenafil attenuated the hypoxia-induced downregulation of PPARγ expression and inhibited the hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. (A) HPASMCs were identified by immunofluorescence staining for α-SMA in cells grown under normoxic conditions. (B) Western blot and (C) RT-qPCR analysis of the increase in PPARγ protein and mRNA expression in HPASMCs induced by different concentrations of sildenafil under hypoxic conditions. Each of the four groups were treated with 0, 1, 10 or 50 nM sildenafil. (D) Western blot and (E) RT-qPCR analysis of the sildenafil-mediated attenuation of hypoxia-induced downregulation of PPARγ expression and sildenafil-mediated inhibition of hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. There were three experimental groups: i) The normoxic control group (60 h), ii) the hypoxia group (60 h, 4% O 2 ), and iii) the hypoxia + sildenafil group (60 h, 4% O 2 , 50 nM sildenafil). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; SMA, smooth muscle actin; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Inhibition, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: A PPARγ inhibitor (GW9662) inhibited the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the inhibitory effects of different concentrations of a PPARγ inhibitor (GW9662) on PPARγ protein expression in HPASMCs under hypoxic conditions. Cells were treated with either 0, 1 or 10 nM GW9662. (C) Western blot and (D) RT-qPCR analysis of the inhibitory effect of a PPARγ inhibitor (GW9662) on the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + GW9662 group (60 h, 4% O 2 , 10 nM), and (iv) the hypoxia + sildenafil + GW9662 group (60 h, 4% O 2 , 10 nM). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control, # P>0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: si-PPARγ reversed the sildenafil-induced downregulation of TRPC and Ki67 expression under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the downregulation of PPARγ protein expression in HPASMCs transfected with si-PPARγ under normoxic conditions. There were four groups: si-NC, siR-PP1, siR-PP2 and siR-PP3. (C) Western blot and (D) RT-qPCR analysis of the reversal of the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs induced by PPARγ siRNA under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + siR-PP1 group (60 h, 4% O 2 ), and iv) the hypoxia + sildenafil + siR-PP1 group. Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; siRNA, small interfering RNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Standard Deviation, Small Interfering RNA, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Respiratory Research

Article Title: STAT6 deficiency mitigates the severity of pulmonary arterial hypertension caused by chronic intermittent hypoxia by suppressing Th2-inducing cytokines

doi: 10.1186/s12931-024-03062-z

Figure Lengend Snippet: IL-4 promotes the proliferation of hPASMCs. The concentration of IL-4 ( A ) and IL-13 ( B ) in serum from STAT6 -/- and WT mice after 6 weeks of normoxia or CIH exposure ( n = 6 per group). The concentration of IL-4 ( C ) and IL-13 ( D ) in BALF from STAT6 -/- and WT mice after 6 weeks of normoxia or CIH exposure ( n = 6 per group). ( E ) CCK8 analysis of hPASMCs with IL-4 treatment under concentration of 0, 0.5, 1,10, and 100 ng/ml. ( F ) CCK8 analysis of hPASMCs with IL-13 treatment under concentration of 0, 0.5, 1,10, and 100 ng/ml. ( G ) The percentage of Ki67-positive cells in 10ng/ml IL-13 or IL-4 treated hPASMCs. Data are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *( p < 0.05), **( p < 0.01), ***( p < 0.001) and determined by Student’s t-test unless specified. hPASMCs: human pulmonary artery smooth muscle cells; Nor: normoxic; CIH: chronic intermittent hypoxia; WT: wide type; BALF: bronchoalveolar lavage fluid

Article Snippet: hPASMCs were obtained from iCell Bioscience, Inc., Shanghai.hPASMCs were cultured at 37 °C in a humidified incubator with 5% CO 2 using PriMed-iCell-004-LS medium (iCell Bioscience, China).Cells at passages 2–5 were used for further investigation.

Techniques: Concentration Assay

Journal: Respiratory Research

Article Title: STAT6 deficiency mitigates the severity of pulmonary arterial hypertension caused by chronic intermittent hypoxia by suppressing Th2-inducing cytokines

doi: 10.1186/s12931-024-03062-z

Figure Lengend Snippet: IL-4 increased p-STAT6 expression in hPASMCs. ( A ) Western blotting was performed to assess the expression of the STAT6 and p-STAT6 in hPASMCs treated with 10ng/ml IL-4 or IL-13 for 24 h. ( B ) Quantitative analysis of relative expression analysis of STAT6 and p-STAT6 using ImageJ. ( C ) Western blotting was used to detect the expression of STAT6 and p-STAT6 in hPASMCs. ( D - E ) Quantitative analysis of STAT6 and p-STAT6 in hPASMCs using ImageJ. ( F ) Under Nor or IH conditions, hPASMCs were treated with 10 ng/ml IL-4 for 48 h. Cell proliferation was assessed using the CCK8 assay. ( G - H ) Knockdown or overexpression of STAT6 expression in hPASMCs, treat with 10 ng/ml IL-4 and culture for 48 h, then measure hPASMCs proliferation under different conditions using the CCK8 assay. Data are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *( p < 0.05), **( p < 0.01), ***( p < 0.001) and determined by Student’s t-test unless specified. Nor normoxia, IH intermittent hypoxia

Article Snippet: hPASMCs were obtained from iCell Bioscience, Inc., Shanghai.hPASMCs were cultured at 37 °C in a humidified incubator with 5% CO 2 using PriMed-iCell-004-LS medium (iCell Bioscience, China).Cells at passages 2–5 were used for further investigation.

Techniques: Expressing, Western Blot, CCK-8 Assay, Knockdown, Over Expression