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Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia

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ABSTRACT

Background: Adipose-derived MSC (AMSCs) possess angiogenic and immunomodulatory properties that may modulate kidney regeneration. Whether these properties are retained in older patients with atherosclerotic vascular disease is poorly understood. Hypoxic conditions are known to modify properties and growth characteristics of AMSCs. We tested the hypothesis that AMSCs from older patients with atherosclerotic renovascular disease (RVD) differ from normal kidney donors, and whether hypoxia changes their functional and molecular properties to promote angiogenesis.

Methods: AMSCs from 11 patients with RVD (mean age =74.5 years) and 10 healthy kidney donors (mean age = 51.2 years) were cultured under normoxia (20 % O2) and hypoxia (1 % O2) for 3–4 days until they reached 80 % confluency. We analyzed expression of genes and microRNAs using RNA sequencing and real-time quantitative rt-PCR. Protein expression of selected angiogenic factors (VEGF, IGF, HGF and EGF) were quantified in conditioned media using ELISAs. Apoptosis was tested using Annexin IV staining.

Results: Normoxic AMSC from RVD patients grew normally, but exhibited increased DNA damage and reduced migration. VEGF protein secretion was significantly lower in the RVD AMSCs (0.08 vs 2.4 ng/mL/ cell, p <0.05) while HGF was higher. Both trends were reversed during growth under hypoxic conditions. Hypoxia upregulated pro-angiogenic mRNAs expression in AMSCs (VEGF, FGF, STC and ANGPTL4), and downregulated expression of many miRNAs (e.g., miR-15a, miR-16, miR-93, miR-424, 126, 132, 221) except miR-210.

Conclusions: Thus, although AMSC from patients with RVD had increased DNA damage and reduced migration, hypoxia stimulated pro-angiogenic responses via increased expression of angiogenic genes, VEGF secretion and induction of the hypoxia-inducible miR-210, while downregulating angiogenesis-related miRNAs.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0389-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Upregulation of gene expression of AMSCs under hypoxia in both RVD and healthy individuals: hypoxia upregulated pro-survival genes (CDKN1A, HMOX1, TMEM45A, CA9, SLC2A1/ GLUT1, and the autophagy regulatory gene BNIP3) (a). Hypoxia upregulated pro-angiogenic genes (ANGPTL4 and VEGF-a) (b) and (STC1, STC2, and FGF2) (c). RVD renovascular disease
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Fig3: Upregulation of gene expression of AMSCs under hypoxia in both RVD and healthy individuals: hypoxia upregulated pro-survival genes (CDKN1A, HMOX1, TMEM45A, CA9, SLC2A1/ GLUT1, and the autophagy regulatory gene BNIP3) (a). Hypoxia upregulated pro-angiogenic genes (ANGPTL4 and VEGF-a) (b) and (STC1, STC2, and FGF2) (c). RVD renovascular disease

Mentions: Expression of more than 3050 mRNAs (out of 23,398 mRNAs) was different in RVD AMSCs vs. healthy volunteers (fold change >1.4; p values <0.05; RVD vs. healthy volunteers). Expression of more than 460 genes with adequate basal levels (RPKM >0.5) was increased under hypoxic conditions (fold change >1.4; p values <0.05; hypoxia vs. normoxia) This set of mRNAs encode regulators of metabolism, DNA repair, cell cycle regulation, and proteins involved in related functions associated with cell survival (Additional file 2: Figure S2). Among these were Cyclin-dependent kinase inhibitor 1A (CDKN1A), TMEM45A, Nox4, BNIP3, SLC2A1 or glucose transporter 1 (GLUT1), carbonic anhydrase 9 (CA 9) and heme oxygenase 1 (HMOX1) genes associated with increased cell survival and anti-apoptotic roles that were highly expressed under hypoxia in both healthy and RVD AMSCs (p <0.05) (Fig. 3a). Hypoxia decreased the expression of protein tyrosine phosphatase, non-receptor type 2 (PTPN2) that negatively regulates numerous signaling pathways and biological processes including cell proliferation and cell growth. Hypoxia increased the expression of BNIP3 and ATG9b, which are known to induce mitochondrial autophagy as well as the pro-angiogenic genes VEGF, FGF, Stanniocalcin-1, -2 (STC 1, 2) and angiopoietin-like 4 (ANGPTL4) (p <0.05) (Fig. 3b,c) both in RVD and healthy donors but to a higher degree in RVD. HGF expression levels were lower under hypoxia in RVD and healthy AMSCs. These data indicate that hypoxia modulates the biological functions of AMSCs and expression of a number of paracrine factors.Fig. 3


Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia
Upregulation of gene expression of AMSCs under hypoxia in both RVD and healthy individuals: hypoxia upregulated pro-survival genes (CDKN1A, HMOX1, TMEM45A, CA9, SLC2A1/ GLUT1, and the autophagy regulatory gene BNIP3) (a). Hypoxia upregulated pro-angiogenic genes (ANGPTL4 and VEGF-a) (b) and (STC1, STC2, and FGF2) (c). RVD renovascular disease
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016873&req=5

Fig3: Upregulation of gene expression of AMSCs under hypoxia in both RVD and healthy individuals: hypoxia upregulated pro-survival genes (CDKN1A, HMOX1, TMEM45A, CA9, SLC2A1/ GLUT1, and the autophagy regulatory gene BNIP3) (a). Hypoxia upregulated pro-angiogenic genes (ANGPTL4 and VEGF-a) (b) and (STC1, STC2, and FGF2) (c). RVD renovascular disease
Mentions: Expression of more than 3050 mRNAs (out of 23,398 mRNAs) was different in RVD AMSCs vs. healthy volunteers (fold change >1.4; p values <0.05; RVD vs. healthy volunteers). Expression of more than 460 genes with adequate basal levels (RPKM >0.5) was increased under hypoxic conditions (fold change >1.4; p values <0.05; hypoxia vs. normoxia) This set of mRNAs encode regulators of metabolism, DNA repair, cell cycle regulation, and proteins involved in related functions associated with cell survival (Additional file 2: Figure S2). Among these were Cyclin-dependent kinase inhibitor 1A (CDKN1A), TMEM45A, Nox4, BNIP3, SLC2A1 or glucose transporter 1 (GLUT1), carbonic anhydrase 9 (CA 9) and heme oxygenase 1 (HMOX1) genes associated with increased cell survival and anti-apoptotic roles that were highly expressed under hypoxia in both healthy and RVD AMSCs (p <0.05) (Fig. 3a). Hypoxia decreased the expression of protein tyrosine phosphatase, non-receptor type 2 (PTPN2) that negatively regulates numerous signaling pathways and biological processes including cell proliferation and cell growth. Hypoxia increased the expression of BNIP3 and ATG9b, which are known to induce mitochondrial autophagy as well as the pro-angiogenic genes VEGF, FGF, Stanniocalcin-1, -2 (STC 1, 2) and angiopoietin-like 4 (ANGPTL4) (p <0.05) (Fig. 3b,c) both in RVD and healthy donors but to a higher degree in RVD. HGF expression levels were lower under hypoxia in RVD and healthy AMSCs. These data indicate that hypoxia modulates the biological functions of AMSCs and expression of a number of paracrine factors.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Adipose-derived MSC (AMSCs) possess angiogenic and immunomodulatory properties that may modulate kidney regeneration. Whether these properties are retained in older patients with atherosclerotic vascular disease is poorly understood. Hypoxic conditions are known to modify properties and growth characteristics of AMSCs. We tested the hypothesis that AMSCs from older patients with atherosclerotic renovascular disease (RVD) differ from normal kidney donors, and whether hypoxia changes their functional and molecular properties to promote angiogenesis.

Methods: AMSCs from 11 patients with RVD (mean age =74.5&nbsp;years) and 10 healthy kidney donors (mean age&thinsp;=&thinsp;51.2&nbsp;years) were cultured under normoxia (20&nbsp;% O2) and hypoxia (1&nbsp;% O2) for 3&ndash;4 days until they reached 80&nbsp;% confluency. We analyzed expression of genes and microRNAs using RNA sequencing and real-time quantitative rt-PCR. Protein expression of selected angiogenic factors (VEGF, IGF, HGF and EGF) were quantified in conditioned media using ELISAs. Apoptosis was tested using Annexin IV staining.

Results: Normoxic AMSC from RVD patients grew normally, but exhibited increased DNA damage and reduced migration. VEGF protein secretion was significantly lower in the RVD AMSCs (0.08 vs 2.4&nbsp;ng/mL/ cell, p &lt;0.05) while HGF was higher. Both trends were reversed during growth under hypoxic conditions. Hypoxia upregulated pro-angiogenic mRNAs expression in AMSCs (VEGF, FGF, STC and ANGPTL4), and downregulated expression of many miRNAs (e.g., miR-15a, miR-16, miR-93, miR-424, 126, 132, 221) except miR-210.

Conclusions: Thus, although AMSC from patients with RVD had increased DNA damage and reduced migration, hypoxia stimulated pro-angiogenic responses via increased expression of angiogenic genes, VEGF secretion and induction of the hypoxia-inducible miR-210, while downregulating angiogenesis-related miRNAs.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0389-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus