Division of Nephrology and Hypertension, Mayo Clinic, 200, First Street SW, Rochester, MN, 55902, USA.
Department of Biochemistry and Molecular Biology, Rochester, USA.
Stem Cell Res Ther. 2021 Apr 14;12(1):240. doi: 10.1186/s13287-021-02310-z.
Atherosclerotic renal artery stenosis (ARAS) is a risk factor for ischemic and hypertensive kidney disease (HKD) for which autologous mesenchymal stem cell (MSC) appears to be a promising therapy. However, MSCs from ARAS patients exhibit impaired function, senescence, and DNA damage, possibly due to epigenetic mechanisms. Hypoxia preconditioning (HPC) exerts beneficial effects on cellular proliferation, differentiation, and gene and protein expression. We hypothesized that HPC could influence MSC function and senescence in ARAS by epigenetic mechanisms and modulating gene expression of chromatin-modifying enzymes.
Adipose-derived MSC harvested from healthy control (N = 8) and ARAS (N = 8) pigs were cultured under normoxia (20%O) or hypoxia (1%O) conditions. MSC function was assessed by migration, proliferation, and cytokine release in conditioned media. MSC senescence was evaluated by SA-β-gal activity. Specific pro-angiogenic and senescence genes were assessed by reverse transcription polymerase chain reaction (RT-PCR). Dot blotting was used to measure global genome 5-hydroxymethylcytosine (5hmC) levels on DNA and Western blotting of modified histone 3 (H3) proteins to quantify tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues.
Specific pro-angiogenic genes in ARAS assessed by RT-PCR were lower at baseline but increased under HPC, while pro-senescence genes were higher in ARAS at baseline as compared healthy MSCs. ARAS MSCs under basal conditions, displayed higher H3K4me3, H3K27me3, and 5hmC levels compared to healthy MSCs. During HPC, global 5hmC levels were decreased while no appreciable changes occurred in histone H3 tri-methylation. ARAS MSCs cultured under HPC had higher migratory and proliferative capacity as well as increased vascular endothelial growth factor and epidermal growth factor expression compared to normoxia, and SA-β-gal activity decreased in both animal groups.
These data demonstrate that swine ARAS MSCs have decreased angiogenesis and increased senescence compared to healthy MSCs and that HPC mitigates MSC dysfunction, senescence, and DNA hydroxymethylation in ARAS MSC. Thus, HPC for MSCs may be considered for their optimization to improve autologous cell therapy in patients with nephropathies.
动脉粥样硬化性肾动脉狭窄（ARAS）是缺血性和高血压性肾病（HKD）的危险因素，自体间充质干细胞（MSC）似乎是一种有前途的治疗方法。然而，来自 ARAS 患者的 MSC 表现出功能障碍、衰老和 DNA 损伤，这可能是由于表观遗传机制。低氧预处理（HPC）对细胞增殖、分化以及基因和蛋白质表达具有有益的影响。我们假设 HPC 可以通过表观遗传机制和调节染色质修饰酶的基因表达来影响 ARAS 中的 MSC 功能和衰老。
从健康对照（N=8）和 ARAS（N=8）猪中分离出脂肪源性 MSC，在常氧（20%O）或低氧（1%O）条件下培养。通过迁移、增殖和条件培养基中细胞因子的释放来评估 MSC 功能。通过 SA-β-半乳糖酶活性评估 MSC 衰老。通过逆转录聚合酶链反应（RT-PCR）评估特定的促血管生成和衰老基因。使用斑点印迹法测量 DNA 上的全基因组 5-羟甲基胞嘧啶（5hmC）水平，并使用 Western blot 检测修饰的组蛋白 3（H3）蛋白以定量三甲基化赖氨酸-4（H3K4me3）、赖氨酸-9（H3K9me3）和赖氨酸-27（H3K27me3）残基。
RT-PCR 评估的 ARAS 中特定的促血管生成基因在基线时较低，但在 HPC 下增加，而促衰老基因在 ARAS 中高于健康 MSC。与健康 MSC 相比，ARAS MSC 在基础条件下显示出更高的 H3K4me3、H3K27me3 和 5hmC 水平。在 HPC 期间，全基因组 5hmC 水平降低，而组蛋白 H3 三甲基化没有明显变化。与常氧相比，在 HPC 下培养的 ARAS MSC 具有更高的迁移和增殖能力，以及更高的血管内皮生长因子和表皮生长因子表达，并且两组的 SA-β-半乳糖酶活性均降低。
这些数据表明，与健康 MSC 相比，猪 ARAS MSC 的血管生成减少，衰老增加，而 HPC 减轻了 ARAS MSC 的 MSC 功能障碍、衰老和 DNA 羟甲基化。因此，对于 MSC 的 HPC 可以考虑对其进行优化，以改善患有肾病的患者的自体细胞治疗。
