immunomagnetic-isolation-vascular-wall-resident-cd34-stem-cells-from

Immunomagnetic Isolation of the Vascular Wall-Resident CD34<sup>+</sup> Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

southwest medical university

Mitochondria play a pivotal role in cellular function, not only acting as the powerhouse of the cell, but also regulating ATP synthesis, reactive oxygen species (ROS) production, intracellular Ca cycling, and apoptosis. During the past decade, extensive progress has been made in the technology to assess mitochondrial functions and accumulating evidences have shown that mitochondrial dysfunction is a key pathophysiological mechanism for many diseases including cardiovascular disorders, such as ischemic heart disease, cardiomyopathy, hypertension, atherosclerosis, and hemorrhagic shock. The advances in methodology have been accelerating our understanding of mitochondrial molecular structure and function, biogenesis and ROS and energy production, which facilitates new drug target identification and therapeutic strategy development for mitochondrial dysfunction-related disorders. This review will focus on the assessment of methodologies currently used for mitochondrial research and discuss their advantages, limitations and the implications of mitochondrial dysfunction in cardiovascular disorders.

Assessment of mitochondrial dysfunction and implications in cardiovascular disorders.

Mitochondrial dysfunction in the endothelium contributes to the progression of hypertension and plays an obligatory role in modulating vascular tone. Acacetin is a natural flavonoid compound that has been shown to possess multiple beneficial effects, including vasodilatation. However, whether acacetin could improve endothelial function in hypertension by protecting against mitochondria-dependent apoptosis remains to be determined. The mean arterial pressure (MAP) in Wistar Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) administered with acacetin intraperitoneally for 2 h or intragastrically for six weeks were examined. The endothelial injury was evaluated by immunofluorescent staining and a transmission electron microscope (TEM). Vascular tension measurement was performed to assess the protective effect of acacetin on mesenteric arteries. Endothelial injury in the pathogenesis of SHR was modeled in HUVECs treated with Angiotensin II (Ang II). Mitochondria-dependent apoptosis, the opening of Mitochondrial Permeability Transition Pore (mPTP) and mitochondrial dynamics proteins were determined by fluorescence activated cell sorting (FACS), immunofluorescence staining and western blot. Acacetin administered intraperitoneally greatly reduced MAP in SHR by mediating a more pronounced endothelium-dependent dilatation in mesenteric arteries, and the vascular dilatation was reduced remarkably by NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis. While acacetin administered intragastrically for six weeks had no apparent effect on MAP, it improved the endothelium-dependent dilatation in SHR by activating the AKT/eNOS pathway and protecting against the abnormalities of endothelium and mitochondria. Furthermore, acacetin remarkably inhibited Ang II induced apoptosis by inhibiting the increased expression of Cyclophilin D (CypD), promoted the opening of mPTP, ROS generation, ATP loss and disturbance of dynamin-related protein 1 (DRP1)/optic atrophy1 (OPA1) dynamics in HUVECs. This study suggests that acacetin protected against endothelial dysfunction in hypertension by activating the AKT/eNOS pathway and modulating mitochondrial function by targeting mPTP and DRP1/OPA1-dependent dynamics.

Restoration of Mitochondrial Function Is Essential in the Endothelium-Dependent Vasodilation Induced by Acacetin in Hypertensive Rats.

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34 VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34 VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34 VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca release and extracellular Ca entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34 VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34 VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca release from endoplasmic reticulum of CD34 VW-SCs. Store-operated Ca entry (SOCE) was activated by using thapsigargin (TG) applied in Ca-free/Ca reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34 VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.

[Isolation, culture and validation of CD34 vascular wall-resident stem cells from mice].

Extracellular adenosine triphosphate (ATP) is one of the most abundant biochemical constitutes within the stem cell microenvironment and is postulated to play critical roles in cell migration. However, it is unclear whether ATP regulates the cell migration of CD34 vascular wall-resident stem/progenitor cells (VW-SCs) and participates in angiogenesis. Therefore, the biological mechanisms of cell migration mediated by ATP was determined by in vivo subcutaneous matrigel plug assay, ex vivo aortic ring assay, in vitro transwell migration assay, and other molecular methods. In the present study, ATP dose-dependently promoted CD34 VW-SCs migration, which was more obviously attenuated by inhibiting or knocking down P2Y2 than P2Y6. Furthermore, it was confirmed that ATP potently promoted the migration of resident CD34 cells from cultured aortic artery rings and differentiation into endothelial cells in matrigel plugs by using inducible lineage tracing -CreER; R26-tdTomato mice, whereas P2Y2 and P2Y6 blocker greatly inhibited the effect of ATP. In addition, ATP enhanced the protein expression of stromal interaction molecule 1 (STIM1) on cell membrane, blocking the calcium release-activated calcium (CRAC) channel with shSTIM1 or BTP2 apparently inhibited ATP-evoked intracellular Ca elevation and channel opening, thereby suppressing ATP-driven cell migration. Moreover, extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 and p38 inhibitor SB203580 remarkably inhibited ERK and p38 phosphorylation, cytoskeleton rearrangement, and subsequent cell migration. Unexpectedly, it was found that knocking down STIM1 greatly inhibited ATP-triggered ERK/p38 activation. Taken together, it was suggested that P2Y2 signaled through the CRAC channel mediated Ca influx and ERK/p38 pathway to reorganize the cytoskeleton and promoted the migration of CD34 VW-SCs. In this study, we observed that the purinergic receptor P2Y2 is critical in the regulation of vascular wall-resident CD34 cells' migration. ATP could activate STIM1-mediated extracellular Ca entry by triggering STIM1 translocation to the plasma membrane, and knockdown of STIM1 prevented ERK/p38 activation-mediated cytoskeleton rearrangement and cell migration.

Qingya Dang

southwest medical university

Immunomagnetic Isolation of the Vascular Wall-Resident CD34⁺ Stem Cells from Mice

Qingya Dang

southwest medical university

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Immunomagnetic Isolation of the Vascular Wall-Resident CD34⁺ Stem Cells from Mice