Journal: International Journal of Molecular Sciences

Article Title: Definition of Synovial Mesenchymal Stem Cells for Meniscus Regeneration by the Mechanism of Action and General Amp1200 Gene Expression

doi: 10.3390/ijms251910510

Figure Lengend Snippet: Cell clustering by Amp1200. Lots of SyMSCs (4 donors: S1, S2, S3 and S4), ADSCs (3 donors: A1, A2 and A3), BMSCs (3 donors: B1, B2 and B3), RECs (3 donors: R1, R2 and R3), FBs, iMSCs, WPs, PAECs, T cells, and CD14+ monocytes were clustered by the expression level of genes obtained by Amp1200. The black lines at the top and left showed the results of the clustering. A similar clustering analysis using all 25,193 human genes is shown in .

Article Snippet: Human FBs (Lonza, Basel, Switzerland, CC-2509), human PAECs (ATCC, Manassas, VA, USA, PCS-100022), human WPs (PromoCell, Heidelberg, Germany, C-12732), human T cells (Lonza, Basel, Switzerland, 2W-200), and human CD14 + monocytes (PromoCell, Heidelberg, Germany, C-12909) were cultured as follows.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Definition of Synovial Mesenchymal Stem Cells for Meniscus Regeneration by the Mechanism of Action and General Amp1200 Gene Expression

doi: 10.3390/ijms251910510

Figure Lengend Snippet: Comparison of R 2 and detection power of the Amp1200-selected, all-human, and random-human gene sets in SyMSCs and different cell types.

Article Snippet: Human FBs (Lonza, Basel, Switzerland, CC-2509), human PAECs (ATCC, Manassas, VA, USA, PCS-100022), human WPs (PromoCell, Heidelberg, Germany, C-12732), human T cells (Lonza, Basel, Switzerland, 2W-200), and human CD14 + monocytes (PromoCell, Heidelberg, Germany, C-12909) were cultured as follows.

Techniques: Comparison

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A, B) Lung sections of mice conditionally lacking Egfr in ECs were stained for CD68 (green), iNOS (white, A) and Arg1 (red, B). (A, B) The numbers of iNOS + (A) and Arg1 + (B) macrophages were quantified by confocal imaging. (C, D, E) Human pulmonary arterial endothelial cells (PAECs) were transfected with either scrambled siRNA (si CTL ) and siRNA against AREG (si AREG ) and placed in a transwell chamber. They were then cultured in hypoxic conditions with or without leukocytes for 24 h. (C) AREG expression was assessed in leukocytes and HPAECs by qPCR. (D) Apoptosis of normoxic and hypoxic HPAECs was quantified by flow cytometry and shown as fold change compared with the level of apoptosis in si CTL PAECs. (E) HPAECs were plated on Matrigel, and tube formation was measured. (F) Human PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. PAECs apoptosis was assessed by measuring caspase 3 + cells and caspase 3 MFI by flow cytometry. Isotype control was used to determine caspase 3 positivity. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Staining, Imaging, Transfection, Cell Culture, Expressing, Flow Cytometry, Recombinant

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: , (A) Schematic representing AREG and its upstream genes including BRCA1 , HLX , NCOA6 , PHB2 , RRP1B , TAF4 , TP63 , and VAV2 was generated using the Ingenuity Pathway Analysis Software. Each arrow represents the activation of AREG by each gene. (B) Schematic depicting HIF-1⍺–binding sites in PHB2 , RRP1B , and NCOA6 gene promoter regions. This schematic was designed using UCSC Genome Browser website ( https://genome.ucsc.edu ) and Snapgene software ( https://www.snapgene.com ). (C, D, E) Pulmonary arterial endothelial cells (PAECs) were transfected with scrambled siRNA (si CTL ) or siRNA against NCOA6 ( siNCOA6 ), PHB2 (si PHB2 ) or RRP1B (si RRP1B ) and placed in normoxia for 24 h. (C) Apoptotic PAECs were quantified by measuring caspase 3 + cells and caspase 3 MFI by flow cytometry. (D) PAECs were plated on Matrigel, and tube formation was assessed. (E) AREG expression was assessed by qPCR. (F) Human PAECs were transfected with either scrambled siRNA (si CTL ), or siRNA against HIF1A and placed in normoxic or hypoxic conditions for 24 h. AREG expression was quantified by qPCR. (G) Human PAECs were transfected with a lentivirus overexpressing HIF1-A for 48 h. The cells were transfected with siRNA against NCOA6 , PHB2 , or RRP1B and cultured in normoxic conditions for 48 h. AREG expression was assessed by qPCR. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Generated, Software, Activation Assay, Binding Assay, Transfection, Flow Cytometry, Expressing, Cell Culture

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A) BAD expression in pulmonary arterial endothelial cells (PAECs) was quantified after AREG silencing (left panel) and recombinant Amphiregulin treatment (right panel). (B, C, F) PAECs were transfected with either scrambled siRNA (si CTL ) or siRNA against BAD (si BAD ) and placed in hypoxia for 24 h. (B, C) PAECs apoptosis was assessed by measuring caspase 3 + and caspase 3 MFI cells by flow cytometry (B), and tube formation ability was determined by a Matrigel assay (C). (D, E) PAECs were transfected with either scrambled siRNA (si CTL ), siRNA against AREG (si AREG ) or siRNA against both AREG and BAD (si BAD/AREG ) and placed in normoxic conditions for 24 h. (D, E) Apoptosis (D) and tube formation (E) were examined. (F) IFNB , IL1B , IL6 , and TNFA expression was assessed by qRT-PCR. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, *** P < 0.005, **** P < 0.001.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Expressing, Recombinant, Transfection, Flow Cytometry, Matrigel Assay, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A) BAD expression and the frequency of BAD + cells were determined by flow cytometry after AREG silencing in normoxic and hypoxic pulmonary arterial endothelial cells (PAECs). (B) PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. BAD expression was measured by RT-qPCR. (C, D) HPAECs were co-cultured in a transwell with leukocytes and then treated with either control or BAD siRNA. (C) Granulocytes, monocytes, and T cells were enumerated by flow cytometry. (D) Cytokine concentrations were assessed by ELISA. (E) Mechanisms of increased PAEC apoptosis and exaggerated inflammation in the absence of AREG and epidermal growth factor receptor (EGFR) in pulmonary hypertension (PH). Our data support a model whereby decreased amphiregulin and EGFR expression in PAECs promote PH. Specifically, in the steady state, amphiregulin binds to the EGFR, which decreases the expression of BCL2-associated agonist of Cell Death (BAD), resulting in PAEC survival and suppressed inflammation. In PH, HIF-1⍺ binds to the promoters of NCOA6 , PHB2 , and RRP1B and increases their expression. These genes down-regulate AREG , resulting in augmented BCL2 expression. This pro-apoptotic gene, in turn, incites apoptosis and chemokine production. Elevated levels of the chemokines recruit inflammatory myeloid cells in lung vasculature. Mechanisms that were not investigated in the present study are labeled with a dotted arrow. The cartoon was designed with the online Biorender software ( https://biorender.com ). n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Expressing, Flow Cytometry, Recombinant, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling, Software

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Western Blot, Infection, BrdU Incorporation Assay

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Endothelial cells characterization regarding morphological, immunophenotyping, and vessel-like structures assay. ( A ) Phase contrast micrography demonstrating the polygonal morphology of aortic artery endothelial cells (PAEC), coronary artery endothelial cells (CAEC), human umbilical vein endothelial cells (HUVEC), and pulmonary artery endothelial cells (HPAEC) cells (100× magnification). ( B ) Immunophenotyping of ECs by flow cytometry. ( C ) All endothelial cells (PAEC, CAEC, HUVEC, and HPAEC) were able to form vessel-like structures when grown in matrigel, evidencing characteristics typical of CEs (40× and 100× magnification).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Characterization of EndMT induction by TGF-β2 (10 ng/mL) in cell lines ( A ) PAEC, ( B ) CAEC, ( C ) HPAEC, and ( D ) HUVECs (non-treated or treated with TGF-β2). Immunofluorescence microscopy of cell lines induced to EndMT shows a decrease in the fluorescent intensity of CD31 (green) in PAECs, CAECs, and HUVECs cells. The nuclei were stained with DAPI (blue) and F-actin were stained with Phalloidin (red) (scale bar 50 µM; representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: TGF-β2 decrease formation of vessel-like structures in the cell lines (CAEC, PAEC, HPAEC, and HUVEC). The cells were treated with TGF-β2 and evaluated the capacity formation of vessel-like structures. This inhibitory effect was observed mainly in PAECs (representative image of one replicate; n = 3).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Effect of EndMT on the activation of the Erk pathway. The cells (CAEC, PAEC, HUVEC and HPAEC) were cultured for five days in presence TGF-β2 (10 ng/mL). Aliquots were withdrawn after the treatment and evaluated by ( A ) Multiplex technique analysis Array Kit ( n = 3, * p ≤ 0.05) and ( B ) western blotting using phospho-Erk1/2 (Thr202/Tyr204) and ERK1/2. β-actin were used as endogenous controls (representative image of one replicate of each sample). ( C ) Chemical inhibitor against MEK1/2 (U0126; 1 μM) inhibits the increase of ERK1/2 phosphorylation in the PAECs treated with TGF-β2. 1) U0126; 2) U0126-15′ TGF-β2; 3) U0126-30′ TGF-β2; 4) TGF-β2-15′; 5) TGF-β2-30′. GAPDH were used as endogenous controls (representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Activation Assay, Cell Culture, Multiplex Assay, Western Blot, Phospho-proteomics