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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

AMSCs from older healthy donors and RVD patients both showed lower secretion of VEGF, IGF and EGF compared to AMSCs from young healthy donors. These data suggest that at least some of the differential effects between healthy donor subjects and RVD are related primarily to differences in age (see text). EGF epidermal growth factor, IGF insulin-like growth factor; HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor
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Fig7: AMSCs from older healthy donors and RVD patients both showed lower secretion of VEGF, IGF and EGF compared to AMSCs from young healthy donors. These data suggest that at least some of the differential effects between healthy donor subjects and RVD are related primarily to differences in age (see text). EGF epidermal growth factor, IGF insulin-like growth factor; HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor

Mentions: Human AMSCs from adipose tissue from both healthy and RVD patients retained characteristic plastic-adherent, fibroblast-like morphology, expressed HLA-ABC, CD44, CD90, CD29, CD73, and CD105 markers, and did not express HLA-DR, CD45, CD14 or CD34 markers either under normoxia or hypoxia (Additional file 1: Figure S1). The morphology of cells grown under hypoxia differed slightly in some individuals (both healthy and RVD), insofar as the cells appeared larger and more granular (Fig. 1). VEGF secretion in the supernatant of RVD AMSC at normoxic conditions was markedly lower compared to healthy controls (p <0.05), whereas HGF levels were slightly higher (Fig. 2a). These changes were mainly due to age rather than vascular disease, as cytokine secretion (IGF, HGF and EGF) was not different in RVD compared to older healthy subjects except for VEGF (Fig. 7). Compared to healthy volunteers (p <0.05), AMSCs from patients with RVD had higher levels of nuclear foci for the phosphorylated form of histone variant H2AX (Fig. 2b). This protein is recruited to sites of DNA damage and phosphorylated by DNA repair kinases, which is particularly evident in aging cells that lose telomeres prior to becoming fully senescent. Under normoxia, cell viability (measured with trypan blue) and percentage of apoptotic cells (measured by Annexin V) were similar in both groups (Fig. 2c). The AMSCs from patients with RVD had lower migration capacity compared to healthy volunteers (Fig. 2d).Fig. 1


Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia
AMSCs from older healthy donors and RVD patients both showed lower secretion of VEGF, IGF and EGF compared to AMSCs from young healthy donors. These data suggest that at least some of the differential effects between healthy donor subjects and RVD are related primarily to differences in age (see text). EGF epidermal growth factor, IGF insulin-like growth factor; HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: AMSCs from older healthy donors and RVD patients both showed lower secretion of VEGF, IGF and EGF compared to AMSCs from young healthy donors. These data suggest that at least some of the differential effects between healthy donor subjects and RVD are related primarily to differences in age (see text). EGF epidermal growth factor, IGF insulin-like growth factor; HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor
Mentions: Human AMSCs from adipose tissue from both healthy and RVD patients retained characteristic plastic-adherent, fibroblast-like morphology, expressed HLA-ABC, CD44, CD90, CD29, CD73, and CD105 markers, and did not express HLA-DR, CD45, CD14 or CD34 markers either under normoxia or hypoxia (Additional file 1: Figure S1). The morphology of cells grown under hypoxia differed slightly in some individuals (both healthy and RVD), insofar as the cells appeared larger and more granular (Fig. 1). VEGF secretion in the supernatant of RVD AMSC at normoxic conditions was markedly lower compared to healthy controls (p <0.05), whereas HGF levels were slightly higher (Fig. 2a). These changes were mainly due to age rather than vascular disease, as cytokine secretion (IGF, HGF and EGF) was not different in RVD compared to older healthy subjects except for VEGF (Fig. 7). Compared to healthy volunteers (p <0.05), AMSCs from patients with RVD had higher levels of nuclear foci for the phosphorylated form of histone variant H2AX (Fig. 2b). This protein is recruited to sites of DNA damage and phosphorylated by DNA repair kinases, which is particularly evident in aging cells that lose telomeres prior to becoming fully senescent. Under normoxia, cell viability (measured with trypan blue) and percentage of apoptotic cells (measured by Annexin V) were similar in both groups (Fig. 2c). The AMSCs from patients with RVD had lower migration capacity compared to healthy volunteers (Fig. 2d).Fig. 1

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