Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Tree dendrogram depicting the relationship between different cell types. Whole-genome bisulfite sequencing (WGBS) data sets were included in the analysis. Average methylation was calculated for each sample within blocks of at least 3 CpGs and the top 30,000 blocks were selected that showed the highest variability across all samples. Unsupervised clustering of the reference WGBS samples was performed based on similarity in methylation status at these highly variable blocks. Samples from cell types with greater than n = 3 replicates were merged. ( B and C ) UMAP plot of human ( B ) and mouse ( C ) WGBS reference data sets. CAEC, coronary artery endothelial cell; CMEC, cardiac microvascular endothelial cell; CPEC, joint cardiopulmonary endothelial cell; HUVEV, human umbilical vein endothelial cell; LSEC, liver sinusoidal endothelial cell; MK, megakaryocyte; NK, natural killer cell; PAEC, pulmonary artery endothelial cell; PMEC, pulmonary microvascular endothelial cell.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Methylation Sequencing, Methylation

Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Functions of genes adjacent to endothelium-specific methylation blocks (all P < 0.05). Blue color indicates nearby hypomethylated regulatory blocks. Yellow color indicates nearby hypermethylated regulatory blocks. ( B ) Example of the NOS3 locus specifically unmethylated in endothelial cells. This endothelium-specific, differentially methylated block (DMB; highlighted in light blue) is 157 bp long (7 CpGs), and is located within the NOS3 gene, an endothelium-specific gene (upregulated in paired RNA-seq data as well as in vascular endothelial cells, GTEx inset). The alignment from the UCSC genome browser (top) provides the genomic locus organization and is aligned with the average methylation (purple tracks) across cardiomyocyte, lung epithelial, liver sinusoidal endothelial cell (LSEC), cardiopulmonary endothelial cell (CPEC), hepatocyte, and immune (PBMC) samples ( n = 3/cell-type group). Results from RNA-seq generated from paired cell types are depicted (green tracks) as well as peak intensity from H3K27ac and H3K4me3 published ChIP-seq data generated in endothelial cells (blue tracks). ( C ) Expression levels of genes adjacent to tissue-specific endothelial methylation blocks. Expression data were generated from paired RNA-seq of the same CPEC and LSEC populations used to generate methylation reference data. Pan-endothelial genes upregulated in both populations (ALL) are identified as common endothelium-specific methylation blocks to both LSEC and CPEC tissue–specific endothelial populations. ( D ) Pathways related to the biological function of genes containing endothelium-specific methylation blocks (all Benjamini-Hochberg–corrected P < 0.05 by right-tailed Fisher’s exact test). Unique pathways to tissue-specific endothelial cells are highlighted in distinct colors. ( E ) Top 5 transcription factor binding sites enriched among endothelium-specific hypomethylated blocks, using HOMER de novo and known motif analysis (cumulative hypergeometric distribution statistic). The background for the HOMER analysis consisted of 3,589 non–endothelial cell-type–specific hypomethylated blocks. HUVEC, human umbilical vein endothelial cell.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Methylation, Blocking Assay, RNA Sequencing Assay, Generated, ChIP-sequencing, Expressing, Binding Assay

Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Representative H&E staining of lung, heart, and liver tissues from mice treated with 3 Gy or 8 Gy radiation compared to sham control. Scale bar: 200 μm. ( B ) qPCR analysis of Cdkn1a (p21) mRNA. The expression in each sample was normalized to Actb and is shown relative to the expression in the sham control. Mean ± SD; n = 3. Kruskal-Wallis test was used for comparisons among groups: lung tissue, P = 0.004; heart tissue, P = 0.025; liver tissue, P = 0.004. ( C ) Lung endothelial, cardiomyocyte, and hepatocyte methylated cfDNA in the circulation of mice treated with 3 Gy and 8 Gy radiation compared to sham control expressed in genome equivalents per mL serum (Geq/mL). cfDNA was extracted from 18 mice ( n = 6 in each group), with cfDNA from 2 mice pooled in each methylome preparation. Mean ± SD; n = 3 independent methylome preparations. Kruskal-Wallis test was used for comparisons among groups. NS, P ≥ 0.05; * P < 0.05: lung endothelial, P = 0.01; cardiomyocyte, P = 0.01; hepatocyte, P = 0.13.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Staining, Control, Expressing, Methylation

Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Representative 3D-CRT treatment planning for patients with left-sided (left) and right-sided (right) breast cancer, respectively. The color map represents different radiation dose levels or isodose lines (green: 95% of prescription dose; isodose lines: yellow = 90%, cyan = 80%, orange = 70%, brown = 50%). ( B , D , and E ) Cardiopulmonary endothelial cell (CPEC), cardiomyocyte, and lung epithelial cfDNA (in Geq/mL) in serum samples. Fragment-level deconvolution used CPEC- ( n = 99), cardiomyocyte- ( n = 374), and lung epithelial cell–specific methylation blocks ( n = 69), respectively. Friedman test compared paired results at baseline, end of treatment (EOT), and recovery time points. * P < 0.05; CPEC P = 0.03, cardiomyocyte P = 0.01, lung epithelial P = 0.99. Mean ± SEM fold change relative to baseline levels is shown in bold ( n = 15). ( C ) Correlation of total endothelial cfDNA with dosimetry data. cfDNA is from deconvolution of pan-endothelial methylation blocks ( n = 131), the mean volume of the lung receiving the 5-Gy dose is represented by Lung V5 Mean (%). Spearman’s correlation r was calculated, and considered significant when * P < 0.05. ( F and G ) Hepatocyte and liver sinusoidal endothelial cell (LSEC) cfDNA (in Geq/mL) in serum samples. Fragment-level deconvolution used hepatocyte ( n = 200) and LSEC methylation blocks ( n = 61). Mean ± SEM fold change relative to baseline levels is shown in bold ( n = 8 right-sided, n = 7 left-sided breast cancer). Wilcoxon’s matched-pairs signed-rank test was used for comparison among groups. * P < 0.05; hepatocyte right-sided P = 0.02, hepatocyte left-sided P = 0.81, LSEC right-sided P = 0.02, and LSEC left-sided P = 0.93.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Methylation, Comparison

Journal: Journal of Cell Science

Article Title: Contribution of protein–protein interactions to the endothelial-barrier-stabilizing function of KRIT1

doi: 10.1242/jcs.258816

Figure Lengend Snippet: Membrane-targeted KRIT1-RE does not rescue endothelial barrier function. (A) Permeability of transduced monolayers to 40 kDa FITC-dextran. Data shown are mean permeability±s.e.m., normalized to scramble shRNA alone (scr), from n =5 independent experiments. (B) TEER of confluent HPAEC monolayers. Resistance reading of an empty FN-coated Transwell was subtracted from resistance values of Transwells containing HPAEC, then multiplied by the growth area of the wells, yielding Ω*cm 2 values. Data shown are mean Ω*cm 2 values±s.e.m., normalized to scramble shRNA alone (negative control). * P <0.05 by Tukey post-hoc testing vs scramble shRNA alone. # P <0.05 by Tukey post-hoc testing vs shKRIT1 alone. P <0.0001 by one-way ANOVA.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC; Cell Applications, Inc., San Diego, CA, Lot #2228) were cultured in Dulbecco's Modified Eagle's Medium DMEM/F-12 (1:1 ratio), supplemented with 5% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (Gibco/Thermo Scientific, Waltham, MA), 1% endothelial cell growth supplement (ECGS; ScienCell, Carlsbad, CA), and 50 μM heparin (Calbiochem, La Jolla, CA).

Techniques: Membrane, Permeability, shRNA, Negative Control

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: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Repurposing rosiglitazone, a PPAR-γ agonist and oral antidiabetic, as an inhaled formulation, for the treatment of PAH

doi: 10.1016/j.jconrel.2018.04.049

Figure Lengend Snippet: The levels of eNOS expression in human PAH PAECs in hypoxic and normoxic condition (A), PPARγ and endothelin-1 in PAH-ECs (B); PPARγ and NOX-4 expression in PAH-SMCs (C). n=3.

Article Snippet: 2.2.10 The effects of rosiglitazone on human PAH-ECs and PAH-SMCs We grew human pulmonary arterials ECs and SMCs (collected from PAH patients), upon receipt from the Pulmonary Hypertension Breakthrough Initiative (PHBI), in gelatin coated dishes using MV2 (PromoCell GmbH, Heidelberg, Germany) and SmGM (Lonza Inc., Williamsport, PA) media, respectively for 5-7 days until their confluency.

Techniques: Expressing