The results of this study identify considerable differences between AMSCs from patients with atherosclerotic RVD and those from healthy controls. Some of these differences were related to older age, but were magnified by disease. Platelet lysate-expanded AMSC from RVD patients exhibited greater levels of senescence-associated DNA damage and reduced migration capacity as compared to AMSC from healthy individuals. Importantly, expression of angiogenic proteins such as VEGF and EGF was reduced in RVD patients, although of HGF levels were higher. Expansion of AMSC from RVD subjects under hypoxic conditions largely reversed the differences seen under normoxic conditions. Hypoxic conditions resulted in a more robust increase in VEGF, which is a key stimulant for angiogenesis. Senescence-associated DNA damage was increased in elderly patients with RVD compared to healthy individuals. Hypoxia enhanced AMSC survival as reflected by a reduced fraction of dead cells in some patients. The angiogenic functions of RVD AMSCs increased to normal levels during hypoxic expansion as reflected by enhanced expression of angiogenesis-related mRNAs (Fig. 3b), alterations in relevant miRNAs and increased secretion of VEGF, IGF and EGF cytokines.
Hypoxic growth has been employed as a strategy to enhance stem cell in vivo survival and tissue regeneration . Our results indicate that these conditions upregulate angiogenic factors and downregulate apoptotic effector pathways. MicroRNAs regulate cellular functions by translational repression and/or mRNA degradation. We focused on common miRNAs known to regulate survival and angiogenesis and their target gene and posttranscriptional protein expressions. Under hypoxia, miR-210 is most consistently and robustly induced . MiR-210 mediates the proliferation and migration under chronic hypoxia and ultimately leads to downregulation of PTPN2. Consistent with previous studies, which showed that inhibition of PTPN2 (a direct target gene of miR-210) lead to increased proliferation and migration of AMSCs , the AMSC from RVD patients under hypoxia in our study highly expressed miR-210 (FC > 65) and downregulated PTPN2.
Our results demonstrate further that hypoxia reduced the expression of miR-34a (FC > -3). Enhanced expression of miR-34a induces apoptosis, cell cycle arrest and differentiation, or reduces migration [30, 31]. Reducing the expression of the pro-apoptotic miR-34a improves survival of bone marrow stem cells in vitro and enhances the therapeutic benefit of cell therapy in mice after acute myocardial infarction . Previous studies have shown that suppression of miR-10a and miR-21 in aged endothelial progenitor cell (EPCs) increase HMGA2 expression, rejuvenate EPCs, resulting in decreased senescence . Hypoxia downregulated both miR-10a (FC > -2) and miR-21 (FC > -3), but without much change in HMGA2 in RVD. CDKN1A is a gene known to regulate cell survival and growth  and is regulated by miR-221-3p and miR-93 . Similarly, our results showed that hypoxia downregulated miR-221 (FC > -2) and miR-93 (FC > -2), CDKN1A was upregulated, thereby potentially promoting survival of AMSCs.
Another important gene overexpressed under hypoxia was HMOX1, which is known to have anti-inflammatory effects and may improve survival in the ischemia/reperfusion-acute kidney injury microenvironment. Liprin-α4, which is required for maintenance of cell-cell contacts , is a hypoxia-inducible gene that was highly expressed in AMSCs under hypoxia (FC >100). TMEM45A has anti-apoptotic functions and is essential for hypoxia-induced protection against apoptosis . We found that TMEM45A was overexpressed under hypoxia in RVD AMSC. Taken together, these changes in mRNA and miRNA indicate that hypoxic conditions boosted cell survival and amplified angiogenic and anti-inflammatory functions of AMSC from atherosclerotic patients.
Autophagy modulates homeostatic and cytoprotective physiological cellular functions, such as degradation of long-lived proteins, organelle turnover, adaptation to stress, extension of lifespan, and cellular development . Recent studies identify crucial functions of BNIP3 and BNIP3L in hypoxia-induced autophagy and indicate that hypoxia impacts cell survival. BNIP3 family disrupts the Bcl-2/Beclin1 complex and induces autophagy, especially when Bcl-2 or Bcl-XL is weakly expressed [39, 40]. In our study, BNIP3 was highly expressed under hypoxia (FC >7), while Bcl-2, Bcl-KL and Beclin1 were downregulated. We suggest that hypoxia-induced autophagy may promote survival, block induction of apoptosis and reduce hypoxic cellular injury, because survival rates between cells grown under normoxia versus hypoxia were comparable, while hypoxia reduced the percentage of dead cells in most RVD patients.
Angiogenesis potentially includes restoration of normal vascular function and structure, and the reversal of vascular senescence. Growth of new blood vessels is crucial in the treatment of RVD and other ischemic diseases [
]. Recent experimental studies indicate that renal VEGF levels are altered in pathologic situations, such as chronic and acute renal ischemia [
]. Our results indicate that AMSC from patients with RVD secreted less VEGF compared to healthy individuals in the AMSC supernatant (Fig.
). Increased expression of angiogenic factors, including the upregulation of VEGF, has previously been shown to increase blood vessel formation in vivo [
]. Elevated expression of miR-210 induces angiogenesis and is associated with local increased VEGF levels [
]. Reduction of miR-15b and miR-16 contributes to an increase in VEGF and improve angiogenesis [
]. Stanniocalcin-1 and -2 (STC 1, 2) promote angiogenic sprouting in human umbilical vein endothelial cells (HUVECs) via VEGF/VEGFR2 and angiopoietin signaling pathways [
]. Consistent with these data, we observed that hypoxia downregulated miR-101b and miR-29a and increased the expression of their target genes STC1 and STC2 in AMSCs.
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
Recent studies show that human miR-221 and miR-222 are important in vascular biology and exhibit marked antiangiogenic properties. They are upregulated in early atherosclerosis, causing inhibition of angiogenic recruitment of endothelial cells (ECs). These miRNAs increase endothelial dysfunction and EC apoptosis and negatively regulate VEGF [48, 49]. We showed in this study that hypoxia decreased the expression of miR-221 (FC approximately -2) and miR-222 (FC > -1.4) and increased expression of their known common target gene CDKN1.
HGF is an angiogenic growth factor that plays an important role in angiogenesis. Its level was increased in AMSCs from RVD compared to healthy donors. HGF is thought to increase the collateral vascular growth (arteriogenesis) , although its role is insufficient to protect vascular integrity in RVD. VEGF alone seems to affect capillary angiogenesis more efficiently than collateral growth . Regulation of local HGF production under hypoxia is unclear, with most reports suggesting that it increases under hypoxia [12, 52]. However, our results indicate that HGF was downregulated under hypoxia both relative to gene expression and protein secretion. Our results are supported by the studies by Hayashi and colleagues  who demonstrated that hypoxic treatment of vascular cells downregulated HGF production due to decreased cAMP, consistent with their potential role in the pathophysiology of ischemic diseases.
Taken together, our results demonstrated that growing AMSCs from RVD patients under hypoxic conditions (1 % O2) altered their cellular characteristics, mRNA and protein expression and increased both survival and angiogenic potential. Oxygen concentration in medullary compartments of the kidney is normally low , and in kidneys with severe RVD accompanied by inflammation, the degree and extent of hypoxia increase [6, 31]. These studies suggest that functional responses of AMSC under hypoxic conditions may favorably influence their efficacy for renal repair. These effects were associated with increased secretion of several cytokines crucial for angiogenesis and survival such as VEGF.