Figure 3.

MT attenuated the calcification and senescence of VSMCs by affecting exosomes in a paracrine manner. VSMCs were treated with fresh conditional medium (control), whole MT‐treated VSMCs conditional medium (MT‐VSMCs‐CM), MT‐treated VSMCs exosome‐free conditional medium (MT‐VSMCs‐exo‐free‐CM) or MT‐treated VSMCs exosome with GW4869 conditional medium (MT‐VSMCs‐GW4869‐CM), respectively. All groups were incubated with β‐GP at the same time. A, The protein levels of RUNX2 and p21 were determined by Western blotting. B, ALP activity assays were determined with an ALP measurement kit. C, CCK8 assay was performed to detect cell proliferation. D, Schematic representation of coculture Transwell system to analyse the exosomal paracrine effects of VSMCs. VSMCs in lower chamber incubated with MT and treated with or without GW4869 were cocultured with VSMCs without treatment in the upper chamber in six‐well Transwell units. And the upper chamber VSMCs were used to determine the expression of RUXN2, p21, ALP activities and cell viability. E, Western blotting showed that RUNX2 and p21 increased in the VSMCs from the upper chamber in MT plus GW4869 group compared with MT plus vehicle. F, ALP activity assays showed that MT plus GW4869 group had higher ALP activity compared with MT plus vehicle. G, CCK8 assay showed that MT plus GW4869 treatment decreased the proliferation of senescence VSMCs compared with MT plus vehicle. Three independent experiments were performed, and representative data are shown. The data represent the mean ± SD. NC, normal control. *P < .05, **P < .01