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Using SWATH-MS to unveil hCSCs response to hCM I/R injury paracrine factors

ABSTRACT: Human Cardiac/Progenitod Stem Cells (hCSCs) transplantation is arising as a novel therapy option for acute myocardial infarction (AMI) patients. The majority of the studies reported point to paracrine signaling as the main effector in hCSCs activation and regenerative capacity. However, the mechanisms of action of these cells in response to AMI paracrine context are still poorly understood. In the present study, a quantitative proteomics analysis (SWATH-MS) was employed to investigate hCSC response to paracrine factors released by human cardiomyocytes (hCMs) in response to an in vitro AMI Ischemia/Reperfusion (I/R) injury. A total of 714 proteins were quantified, from which 86 were differentially expressed, including 63 proteins up-regulated and 23 down-regulated in hCSCs exposed to hCM I/R injury conditioned media vs hCSCs exposed to control hCM conditioned media. Bioinformatics functional analysis revealed up-regulation of proteins involved with endocytosis, paracrine signaling, proliferation, migration and stress response. This work provides an insight into hCSC biology in response to paracrine cues derived from hCMs upon I/R injury, and the data generated herein constitutes a rich resource for further studies aiming at potentiating the regenerative properties of these cells.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart, Stem Cell, Cell Culture

SUBMITTER: Maria Sebastião

LAB HEAD: Paula Marques Alves

PROVIDER: PXD011998 | Pride | 2019-09-25

REPOSITORIES: Pride

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			Action	DRS
	LibrarygelIDA.group	Other
	LibrarygelIDA.txt	Txt
	Librarywiff.rar	Other
	PeptideandproteinreportsfortheproteinsquantifiedbySWATH-MSMJS.xlsx	Xlsx
	SWATH_H1_Ctrl_0h_1.wiff	Wiff

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Project description:To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested at 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (p<0.05, FDR<1%) were quantified by SWATH-MS. Bioinformatics data analysis showed that there were 35 up-regulated and 24 down-regulated proteins in the temporal quadrant while 19 and 5 proteins were up-regulated and down-regulated, respectively, in the nasal quadrant respectively (n=4, p<0.05; fold change ≥1.4 fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants including CLU, GFAP, GNG5, IRF2BPL, L1CAM and CPLX1. Linear regression analysis indicated a strong association between the data obtained by SWATH-MS and MRM-MS (temporal, R2=0.97; nasal, R2=0.96). Gene ontology analysis reveals statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

Project description:Rationale and Objectives: Cellular therapies are hampered by poor survival and growth of grafts. We recently showed that the survival and repairing capacity of transplanted adult cells may be improved by co-expression of the anti-senescence telomerase reverse transcriptase (TERT) and anti-apoptotic myocardin (MYOCD). The current study aimed at testing whether forced co-expression of TERT and MYOCD improves post-infarct revascularization and tissue repair by adipose tissue-derived mesenchymal stromal cells (AT-MSCs). Methods: We transplanted AT-MSCs engineered to overexpress MYOCD and TERT in a murine model of acute myocardial infarction (AMI). We analyzed the immunoregulatory properties of AT-MSCs on post-AMI. We characterized in vitro and in vivo paracrine effects of AT-MSCs and their interactions with murine cardiospheres (CSps), all as parts of their reparative repertoire. Results: When transplanted into infarcted hearts of C57BL/6 mice, “rejuvenated” AT-MSCs overexpressing TERT and MYOCD (rAT-MSCs) decreased fibrosis and the intrafibrotic CD3 and B220 lymphocyte infiltration, and increased arteriolar density as well as ejection fraction compared with saline or mock-transduced AT-MSCs. These effects were accompanied by increased numbers of Ki-67+ and CD117+ cells, and the expression of cardiac actin and β-myosin heavy chain within the infarcted hearts. Both rAT-MSCs and their conditioned medium (CM) stimulated intracellular Ca2+ flux in CSps. CSp-derived cells had increased survival when preconditioned with CM or exosome-enriched fraction (Exo+) from rAT-MSCs and then exposed to simulated ischemia/reperfusion. Proteomic analysis of rAT-MSCs-Exo+ predicted the activation of vascular development and the inhibition of immune cell trafficking. Elevated concentration of miR-320a, miR-150-5p and miR-126-3p associated with regulation of apoptosis and vasculogenesis was confirmed in rAT-MSCs-Exo+. Conclusions: rAT-MSCs promote vasculogenesis and cell survival and reduce post-AMI inflammatory responses in a murine model of AMI. An integrated experimental and computational analysis revealed that Exo+ is the active component of the paracrine secretion by rAT-MSCs, with pro-angiogenic and pro-survival activities.

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Using SWATH-MS to unveil hCSCs response to hCM I/R injury paracrine factors

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