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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

Hypoxia changed the expression of miRNAs regulating survival and pro-angiogenic genes: hypoxia upregulated the pro-angiogenic miRNA-210 (65 FC in RVD, but to a lesser degree in healthy control 2.9 FC) (a), whereas downregulated pro-apoptotic miRNAs (miR-34a, miR-15, and miR-16) and also downregulated antiangiogenic miRNAs (miR-221/222 and miR-424) only in RVD (b). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease
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Fig5: Hypoxia changed the expression of miRNAs regulating survival and pro-angiogenic genes: hypoxia upregulated the pro-angiogenic miRNA-210 (65 FC in RVD, but to a lesser degree in healthy control 2.9 FC) (a), whereas downregulated pro-apoptotic miRNAs (miR-34a, miR-15, and miR-16) and also downregulated antiangiogenic miRNAs (miR-221/222 and miR-424) only in RVD (b). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease

Mentions: There were 415 out of 2253 miRNAs that were different in RVD from healthy donors under normal conditions (FC >1.4). We detected more than 360 miRNAs with acceptable basal levels (normalized reads >0.2) and differentially expressed in hypoxia (fold change >1.4; p values <0.05; hypoxia vs. normoxia). The majority of these miRNAs (approximately 85 %) were downregulated under hypoxia, with the most notable exception being the pro-angiogenic miR-210 (that increases VEGF levels), which was highly upregulated (fold change FC >65, in RVD cells). miRNA-210 was also increased in healthy control AMSCs under hypoxia (2.9 FC) but to a lesser degree than was seen in RVD. Baseline miR-210 was low in RVD compared to healthy control AMSCs (Fig. 5a). The miRNAs that exhibited reduced expression under hypoxia, included pro-apoptotic miRNAs (miR-34a, miR-15 and miR-16), senescence-associated miRNAs (miR-10a and miR-21) and anti-angiogenic miRNAs (miR-221/222, miR-24, miR-29, miR-15, miR-16, miR-17, miR-424, miR-20b, miR-218, miR-132, miR-92a, and miR-101) (Fig. 5b). The latter miRNAs may negatively modulate angiogenesis by repressing VEGF expression under normoxic conditions. For example, reduction of miR-15, miR-16, and miR-424 contributes to increased VEGF expression [24–26]. Downregulation of miR-221 correlates with an increase in its target gene expression CDKN1A in our study (Fig. 3a), consistent with previous observations [27]. There were not similar effects of hypoxia on most miRNAs from healthy donors AMSCs ‘both young and old’.Fig. 5


Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia
Hypoxia changed the expression of miRNAs regulating survival and pro-angiogenic genes: hypoxia upregulated the pro-angiogenic miRNA-210 (65 FC in RVD, but to a lesser degree in healthy control 2.9 FC) (a), whereas downregulated pro-apoptotic miRNAs (miR-34a, miR-15, and miR-16) and also downregulated antiangiogenic miRNAs (miR-221/222 and miR-424) only in RVD (b). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016873&req=5

Fig5: Hypoxia changed the expression of miRNAs regulating survival and pro-angiogenic genes: hypoxia upregulated the pro-angiogenic miRNA-210 (65 FC in RVD, but to a lesser degree in healthy control 2.9 FC) (a), whereas downregulated pro-apoptotic miRNAs (miR-34a, miR-15, and miR-16) and also downregulated antiangiogenic miRNAs (miR-221/222 and miR-424) only in RVD (b). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease
Mentions: There were 415 out of 2253 miRNAs that were different in RVD from healthy donors under normal conditions (FC >1.4). We detected more than 360 miRNAs with acceptable basal levels (normalized reads >0.2) and differentially expressed in hypoxia (fold change >1.4; p values <0.05; hypoxia vs. normoxia). The majority of these miRNAs (approximately 85 %) were downregulated under hypoxia, with the most notable exception being the pro-angiogenic miR-210 (that increases VEGF levels), which was highly upregulated (fold change FC >65, in RVD cells). miRNA-210 was also increased in healthy control AMSCs under hypoxia (2.9 FC) but to a lesser degree than was seen in RVD. Baseline miR-210 was low in RVD compared to healthy control AMSCs (Fig. 5a). The miRNAs that exhibited reduced expression under hypoxia, included pro-apoptotic miRNAs (miR-34a, miR-15 and miR-16), senescence-associated miRNAs (miR-10a and miR-21) and anti-angiogenic miRNAs (miR-221/222, miR-24, miR-29, miR-15, miR-16, miR-17, miR-424, miR-20b, miR-218, miR-132, miR-92a, and miR-101) (Fig. 5b). The latter miRNAs may negatively modulate angiogenesis by repressing VEGF expression under normoxic conditions. For example, reduction of miR-15, miR-16, and miR-424 contributes to increased VEGF expression [24–26]. Downregulation of miR-221 correlates with an increase in its target gene expression CDKN1A in our study (Fig. 3a), consistent with previous observations [27]. There were not similar effects of hypoxia on most miRNAs from healthy donors AMSCs ‘both young and old’.Fig. 5

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