Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN mice underwent hindlimb ischemia surgery and 1.5 × 10 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

Background: WNK kinase is a serine/threonine kinase that plays an important role in normal blood pressure homeostasis. WNK3 was previously found to enhance the activity of sodium chloride cotransporter (NCC) in Xenopus oocyte. However, the mechanism through which it works remains unclear. Methods: Using overexpression and siRNA knock-down techniques, the effects of WNK3 on NCC in both Cos-7 and mouse distal convoluted cells were analyzed by Western blot. Results: We found that WNK3 significantly increased NCC protein expression in a dose-dependent manner. NCC protein expression in Cos-7 cells was markedly decreased after 2 h treatment with protease inhibitor, cycloheximide (CHX) in the NCC alone group, but was significantly decreased after 8 h treatment of CHX in the WNK3 + NCC group. WNK3 significantly increased NCC protein expression in both NCC alone and WNK3 + NCC groups regardless the overnight treatments of bafilomycin A1, a proton pump inhibitor, suggesting that WNK3-mediated increased NCC expression is not dependent on the lysosomal pathway. We further found that WNK3 group had a quicker NCC recovery than the control group using CHX pulse assay, suggesting that WNK3 increases NCC protein synthesis. WNK3 enhanced NCC protein level while reducing ERK 1/2 phosphorylation. In addition, knock-down of ERK 1/2 expression reversed WNK3-mediated increase of NCC expression. Conclusion: These results suggest that WNK3 enhances NCC protein expression by increasing NCC synthesis via an ERK 1/2-dependent signaling pathway.

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Ran‐Binding protein M (RanBPM) is a nucleoplasmic protein. A recent study has shown that RanBPM is an inhibitor of ERK 1/2 signaling pathway by regulating c‐Raf stability. WNK is a serine/threonine kinase. Mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type II (PHA II), characterized by hypertension, hyperkalemia and metabolic acidosis. Our previous study has shown that WNK4 inhibits NCC by activating ERK 1/2 signaling pathway. To determine whether WNK4 inhibits NCC through RanBPM‐dependent ERK 1/2 signaling pathway, we first investigated interaction between WNK4 and RanBPM using yeast‐two hybrid screening technique. We found that WNK4 interacted with RanBPM. RanBPM was also shown to prevent ERK 1/2 phosphorylation in response to EGF stimulation. When mouse distal convoluted cells (mDCT) were transfected with myc‐RanBPM, ERK 1/2 phosphorylation was decreased while NCC protein expression increased. When mDCT cells were transfected with siRNA RanBPM, ERK 1/2 phosphorylation was increased. These data suggest that 1) WNK4 interacts with RanBPM which is an inhibitor of ERK 1/2 phosphorylation; 2) RanBPM mediates NCC regulation likely through ERK1/2 signaling pathway.