Further intracellular proteins and signaling pathways regulated by angiotensin-(1–7) in human endothelial cells

In 2016, Meinert et al. (doi: 10.1016/j.jprot.2015.09.020) published the first 25 proteins in a protein array regulated in Human Umbilical Vein Endothelial Cells (HUVEC) by the heptapeptide angiotensin (Ang)-(1–7) and the first 10 intracellular signaling cascades at different time points. This supporting data article shows further proteins and pathways stimulated by Ang-(1–7) in human endothelial cells at time points of 1 h, 3 h, 6 h, and 9 h. HUVECs were stimulated with Ang-(1–7), and regulated proteins were identified via antibody microarray. Bioinformatics software IPA was used for association of regulated proteins to metabolic pathways.


Subject area Cardiovascular
More specific subject area

Renin-angiotensin system
Type of data 5 tables How data was acquired Antibody microarray for regulated proteins using a GenePix 4100A Microarray Scanner (Molecular Devices, Sunnyvale, USA), and the program IPA (Ingenuity Systems, Redwood City, USA) for the identification of potential metabolic pathways.

Data format analyzed Experimental factors
Human Umbilical Vein Endothelial Cells were stimulated with angiotensin-(1-7)

Experimental features
Screening of proteins and pathways in angiotensin-(1-7) stimulated Human Umbilical Vein Endothelial Cells Data source location

Cork, Ireland
Data accessibility Data within this article

Value of the data
First screening of 725 proteins potentially regulated by angiotensin (Ang)-(1-7) in endothelial cells via antibody microarray.
As often slightly regulated proteins have already dramatic biological effects, identification of further proteins altered by Ang-(1-7) might have significant scientific relevance.
Detailed description of Ang-(1-7) effects on intracellular signaling pathways under nonpathophysiological circumstances can identify further areas of benefit using Ang-(1-7).
The understanding of intracellular network signaling initiated by Ang-(1-7) might allow conclusions on how the heptapeptide can oppose the effects of the detrimental Ang II.

Data
The antibody microarray identified 110 regulated proteins in human umbilical vein endothelial cells (HUVEC) cells after 1-h stimulation with Ang-(1-7), 119 after 3 h, 31 after 6 h, and 86 after 9 h. The first 25 regulated proteins have been published in Meinert et al. [1] in Tables 1-4. Here the name and ranking of the next regulated proteins are shown (Tables 1-4). Additionally, further intracellular pathways affected by Ang-(1-7) are shown in Table 5A-D.

Cell culture and cell stimulation
HUVEC were grown on 100-mm dishes in EBM (Endothelial basal medium)-2 medium under standard conditions of 37°C in a humidified incubator and 5% CO 2 [2]. Cells were used in passage 6. When they reached 70% confluence, they were washed twice with DPBS (Dulbecco's phosphatebuffered saline) and serum starved for 1 h in supplements-free medium. HUVECs were stimulated with 10 À 7 M Ang-(1-7) for 1 h, 3 h, 6 h and 9 h. Control cells were treated only with DPBS (solvent). Table 1 The proteins ranked 26-100 based on the detected fold change values after 1 h incubation of HUVEC with 10 À 7 M Ang-(1-7). The order of the numbers is oriented on the highest single value. Expression fold change lower than 1.5 is given in hyphen. Data that could not be detected is marked as n.d. Proteins marked in Italic show repeatedly identified differentially expressed proteins (RIDEPs). The mentioned dye indicates with which dye the unstimulated sample was labeled with.

Protein
Antibody  Table 2 The proteins ranked 26-100 based on the detected fold change values after 3 h incubation of HUVEC with 10 À 7 M Ang-(1-7). The order of the numbers is oriented on the highest single value. Expression fold change lower than 1.5 is given in hyphen. Data that could not be detected is marked as n.d. Proteins marked in Italic show repeatedly identified differentially expressed proteins (RIDEPs). The mentioned dye indicates which dye the unstimulated sample was labeled with.   Table 3 The proteins ranked 26-31 based on the detected fold change values after 6 h incubation of HUVEC with 10 À 7 M Ang- (1-7). The order of the numbers is oriented on the highest single value. Expression fold change lower than 1.5 is given in hyphen. Data that could not be detected is marked as n.d. Proteins marked in Italic show repeatedly identified differentially expressed proteins (RIDEPs). The mentioned dye indicates which dye the unstimulated sample was labeled with.   Table 5 Metabolic pathways ranked position 11-25 (bold) using the p-values associated by the IPA software to each of the different antibody microarray sets (A: 1 h; B: 3 h; C: 6 h; D: 9 h). For completion, the ranking of the first ten pathways are also listed (in Italic). The ratio states the number of proteins detected in the microarray versus the total number of proteins being part of the particular pathway.

Antibody microarray
After Ang-(1-7) stimulation, 1 mg/ml protein cell extract was labeled with Cy™3 or Cy™5 dye. The antibody microarray was performed as described in the Panorama Antibody Microarray-XPRESS Profiler725 Kit manual (Sigma-Aldrich, St. Louis, USA). After incubation with the labeled samples, washing and air drying images were acquired using GenePix 4100A Microarray Scanner (Molecular Devices, Sunnyvale, USA). Data was imported into Acuity 4.0 software (Molecular Devices, Sunnyvale, USA) and normalized using the nonlinear Lowess normalization method. Association of regulated proteins to metabolic pathways was done by IPA software (Ingenuity Systems, Redwood City, USA). The software calculated a p-value using the right tailed Fisher Exact test. The p-value gives the probability that the association between regulated detected proteins and the pathways is due to random association. The software considers a p-value o0.05 as statistically significant.