Search

Mitochondria are intracellular organelles that regulate cell survival and death, and hyperglycemia modulates mitochondrial function in endothelial cells. In the current study, we have discovered that high glucose (HG) treatment reduces FUNDC1 (FUN14 domain containing 1) expression in endothelial cells. FUNDC1 expression in mitochondria inhibits proteasomal degradation of COX-IV, and regulates mitochondrial complexes I and IV activity and ATP synthesis. FUNDC1 depletion in HG affects mitochondrial complexes I and IV activity and ATP synthesis and promotes mitochondrial damage through loss of mitochondrial membrane potential and ROS (Reactive Oxygen Species) production. BAM15, a mitochondrial uncoupler, improves mitochondrial function and endothelial survival more effectively. Co-treatment of HG with BAM15 increased FUNDC1 protein expression, mitochondrial translocation of FUNDC1 in HG-treated cells. BAM15-induced up regulation of FUNDC1 expressions improve mitochondrial expression of COX-IV, and complex I and IV activity and ATP synthesis. Our findings propose that FUNDC1 expression in endothelial cells under hyperglycemic stress play a significant role in limiting vascular damage and apoptotic cell death.

Cardiotoxicity caused by doxorubicin (DOX) is an important issue to consider for both patients and doctors who require DOX. DOX-induced cardiotoxicity is closely associated with cardiomyocyte death and dysfunction. To prevent DOX-induced cardiotoxicity, many studies have been conducted on new therapeutic strategies, including the discovery of novel functional modulators such as antioxidant drugs to restore the loss of function of transplanted or residual cardiac cells in the heart. We investigated whether glutathione (GSH), an antioxidant drug, has a protective effect against DOX-induced cardiotoxicity by decreasing ROS and unraveling the underlying molecular mechanisms. GSH clearly increased the viability of damaged human cardiac progenitor cells (hCPCs) treated with DOX. In addition, ROS generation and apoptosis induced by DOX treatment were significantly reduced. We also observed that GSH restored the capacity of hCPCs, as shown by the wound healing assay, transwell migration, and tube formation. We checked that GSH treatment restored the level of pERK, which increased in the DOX-treated group. The ERK inhibitor, U0126, increased the viability of damaged hCPCs. These data suggest that the restoration mechanism of GSH may be via the regulation of pERK signaling. We confirmed the effects of DOX and GSH using an in vivo model. As a result, GSH was confirmed to have a protective effect against DOX-induced cardiotoxicity through body weight, survival rate, histology, and mRNA level. Taken together, GSH prevents DOX-induced cardiotoxicity and regulates pERK signaling. GSH may be an effective therapeutic strategy for DOX-induced cardiotoxicity.

Cardiovascular disease (CVD) and its complications are the leading cause of morbidity and mortality in the world. Because of the side effects and incomplete recovery from current therapy, stem cell therapy emerges as a potential therapy for CVD treatment, and endothelial progenitor cell (EPC) is one of the key stem cells used for therapeutic applications. The effect of this therapy required the expansion of EPC function. To enhance the EPC activation, proliferation, and angiogenesis using dronedarone hydrochloride (DH) is the purpose of this study. DH received approval for atrial fibrillation treatment and its cardiovascular protective effects were already reported. In this study, DH significantly increased EPC proliferation, tube formation, migration, and maintained EPCs surface marker expression. In addition, DH treatment up-regulated the phosphorylation of AKT and reduced the reactive oxygen species production. In summary, the cell priming by DH considerably improved the functional activity of EPCs, and the use of which might be a novel strategy for CVD treatment.

BACKGROUND: Despite promising advances in stem cell-based therapy, the treatment of ischemic cardiovascular diseases remains a big challenge due to both the insufficient in vivo viability of transplanted cells and poor angiogenic potential of stem cells. The goal of this study was to develop therapeutic human cardiac progenitor cells (hCPCs) for ischemic cardiovascular diseases with a novel M13 peptide carrier. METHOD: In this study, an engineered M13 peptide carrier was successfully generated using a QuikChange Kit. The cellular function of M13 peptide carrier-treated hCPCs was assessed using a tube formation assay and scratch wound healing assay. The in vivo engraftment and cell survival bioactivities of transplanted cells were demonstrated by immunohistochemistry after hCPC transplantation into a myocardial infarction animal model. RESULTS: The engineered M13(RGD+SDKP) peptide carrier, which expressed RGD peptide on PIII site and SDKP peptide on PVIII site, did not affect morphologic change and proliferation ability in hCPCs. In contrast, hCPCs treated with M13(RGD+SDKP) showed enhanced angiogenic capacity, including tube formation and migration capacity. Moreover, transplanted hCPCs with M13(RGD+SDKP) were engrafted into the ischemic region and promoted in vivo cell survival. CONCLUSION: Our present data provides a promising protocol for CPC-based cell therapy via short-term cell priming of hCPCs with engineered M13(RGD+SDKP) before cell transplantation for treatment of cardiovascular disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00244-w) contains supplementary material, which is available to authorized users.

Anterior gradient protein 2 homolog (AGR2), an endoplasmic reticulum protein, is secreted in the tumor microenvironment. AGR2 is a member of the disulfide isomerase family, is highly expressed in multiple cancers, and promotes cancer metastasis. In this study, we found that etravirine, which is a non-nucleoside reverse transcriptase inhibitor, could induce AGR2 degradation via autophagy. Moreover, etravirine diminished proliferation, migration, and invasion in vitro. Moreover, in an orthotopic xenograft mouse model, the combination of etravirine and paclitaxel significantly suppressed cancer progression and metastasis. This drug may be a promising therapeutic agent for the treatment of ovarian cancer.

Colorectal cancer is one of the leading causes of cancer-related deaths. Due to relapse after current therapy regimens, cancer stem cells (CSCs) are being studied to target this small tumor-initiating population. Anterior gradient 2 (AGR2), a disulfide isomerase protein, is a well-known pro-oncogenic/metastatic oncogene overexpressed in various tumor tissues, including colon cancer. We found that AGR2 was a novel stem cell marker that was regulated by the canonical Wnt/β-catenin pathway in colon CSCs. AGR2 was highly co-expressed with surface stem cell markers in spheroidal culture. Silencing of AGR2 resulted in decreased sphere-forming ability and down-regulated expression of stem cell markers, whereas the opposite effects were seen with AGR2 overexpression. Moreover, patients with high β-catenin and AGR2 expression showed lower overall survival than those with low expression. In conclusion, our study describes a novel role for AGR2 as a stem cell marker that is highly regulated by canonical Wnt/β-catenin signaling in colorectal cancer.

Anterior gradient protein 2 homolog (AGR2) belongs to the disulfide isomerase family of endoplasmic reticulum proteins. Itis overexpressed in several types of solid tumors, including tumors of the prostate, lung, and pancreas. However, the role of AGR2 in breast cancer and the regulatory mechanisms underlying AGR2 protein expressionare not fullyunderstood. We demonstrated that AGR2 levels are increased under hypoxic conditions and in breast cancer tumors. Mechanistically, Twist1 binds to, and activates the AGR2 promoter via an E-box sequence. Under hypoxic conditions, the increased expression of ARG2 is attenuated when Twist1 levels are reduced by shRNA. Conversely, Twist1 overexpression fully reverses decreased AGR2 levels upon HIF-1α knockdown. Notably, AGR2 is required for Twist1-induced proliferation, migration, and invasion of breast cancer cells. Collectively, these findings extend our understanding of AGR2 regulation in breast cancer and may contribute to development of Twist1-AGR2 targeting therapeutics for breast cancer.

Intercellular interaction is the most crucial factor in promoting cell viability and functionality in an engineered tissue system. Of the various shapes available for cell-laden constructs, spheroidal multicellular microarchitectures (SMMs) have been introduced as building blocks and injectable cell carriers with substantial cell-cell and cell-extracellular matrix (ECM) interactions. Here, we developed a precise and expeditious SMM printing method that can create a tissue-specific microenvironment and thus be potentially useful for cell therapy. This printing strategy is designed to manufacture SMMs fabricated with optimal bioink blended with decellularized ECM and alginate to enhance the functional performance of the encapsulated cells. Experimental results showed that the proposed method allowed for size controllability and mass production of SMMs with high cell viability. Moreover, SMMs co-cultured with endothelial cells promoted lineage-specific maturation and increased functionality compared to monocultured SMMs. Overall, it was concluded that SMMs have the potential for use in cell therapy due to their high cell retention and proliferation rate compared to single-cell injection, particularly for efficient tissue regeneration after myocardial infarction. This study suggests that utilizing microextrusion-based 3D bioprinting technology to encapsulate cells in cell-niche-standardized SMMs can expand the range of possible applications.

Stem cell-based therapies with clinical applications require millions of cells. Therefore, repeated subculture is essential for cellular expansion, which is often complicated by replicative senescence. Cellular senescence contributes to reduced stem cell regenerative potential as it inhibits stem cell proliferation and differentiation as well as the activation of the senescence-associated secretory phenotype (SASP). In this study, we employed MHY-1685, a novel mammalian target of rapamycin (mTOR) inhibitor, and examined its long-term priming effect on the activities of senile human cardiac stem cells (hCSCs) and the functional benefits of primed hCSCs after transplantation. In vitro experiments showed that the MHY-1685‒primed hCSCs exhibited higher viability in response to oxidative stress and an enhanced proliferation potential compared to that of the unprimed senile hCSCs. Interestingly, priming MHY-1685 enhanced the expression of stemness-related markers in senile hCSCs and provided the differentiation potential of hCSCs into vascular lineages. In vivo experiment with echocardiography showed that transplantation of MHY-1685‒primed hCSCs improved cardiac function than that of the unprimed senile hCSCs at 4 weeks post-MI. In addition, hearts transplanted with MHY-1685-primed hCSCs exhibited significantly lower cardiac fibrosis and higher capillary density than that of the unprimed senile hCSCs. In confocal fluorescence imaging, MHY-1685‒primed hCSCs survived for longer durations than that of the unprimed senile hCSCs and had a higher potential to differentiate into endothelial cells (ECs) within the infarcted hearts. These findings suggest that MHY-1685 can rejuvenate senile hCSCs by modulating autophagy and that as a senescence inhibitor, MHY-1685 can provide opportunities to improve hCSC-based myocardial regeneration., Heart disease: keeping cardiac stem cells younger Stem cells for repairing damaged hearts could be kept in a younger and more viable state using a drug called MHY-1685 which assists processes that resist cell aging. Using stem cells to repair damaged heart tissue requires millions of cells but culturing a suitable cell type often causes many cells to age and become less viable. Ji Hye Park at Pusan University, Yangsan, South Korea, and colleagues explored the potential of MHY-1685 to rejuvenate human cardiac stem cells (hCSCs). The drug enhances a maintenance process called autophagy, which clears cells of worn-out components. Exposure to MHY-1685 generated stem cell populations that proved more effective than unexposed cells at repairing damaged heart tissue when transplanted into rats. Its potential for producing cells for treating patients should be explored.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with self-renewing properties; thus, transplanting functionally enhanced MSCs might be a promising strategy for cell therapy against ischemic diseases. However, extensive oxidative damage in ischemic tissue affects the cell fate of transplanted MSCs, eventually resulting in cell damage and autophagic cell death. Oleuropein (OLP) is a bioactive compound isolated from olives and olive oil that harbors antioxidant properties. This study aimed to investigate the potential cytoprotective effects of OLP against oxidative stress and autophagic cell death in MSCs. We found that short-term priming with OLP attenuated H2O2-induced apoptosis by regulating the pro-apoptotic marker Bax and the anti-apoptotic markers Bcl-2 and Mcl-1. Notably, OLP inhibits H2O2 -induced autophagic cell death by modulating autophagy-related death signals, including mTOR (mammalian target of rapamycin), ULK1 (unc-51 like autophagy activating kinase 1), Beclin-1, AMPK (AMP-activated protein kinase), and LC3 (microtubule-associated protein 1a/1b-light chain 3). Our data suggest that OLP might reduce H2O2-induced autophagy and cell apoptosis in MSCs by regulating both the AMPK-ULK axis and the Bcl-2-Mcl-1 axis. Consequently, short-term cell priming with OLP might enhance the therapeutic effect of MSCs against ischemic vascular diseases, which provides an important potential improvement for emerging therapeutic strategies.

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the NF-κB family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity. [BMB Reports 2018; 51(2): 92-97].

Dysfunction or loss of blood vessel causes several ischemic diseases. Although endothelial progenitor cells (EPCs) are a promising source for cell-based therapy, ischemia-induced pathophysiological condition limits the recovery rate by causing drastic cell death. To overcome this issue, we attempted to develop a cell-targeted peptide delivery and priming system to enhance EPC-based neovascularization using an engineered M13 bacteriophage harboring nanofibrous tubes displaying ~2700 multiple functional motifs. The M13 nanofiber was modified by displaying RGD, which is an integrin-docking peptide, on the minor coat protein, and by mutilayering SDKP motifs, which are the key active sites for thymosin β4, on the major coat protein. The engineered M13 nanofiber dramatically enhanced ischemic neovascularization by activating intracellular and extracellular processes such as proliferation, migration, and tube formation in the EPCs. Furthermore, transplantation of the primed EPCs with the M13 nanofiber harboring RGD and SDKP facilitated functional recovery and neovascularization in a murine hindlimb ischemia model. Overall, this study demonstrates the effectiveness of the M13 nanofiber-based novel peptide delivery and priming strategy in promoting EPC bioactivity and neovessel regeneration. To our knowledge, this is first report on M13 nanofibers harboring dual functional motifs, the use of which might be a novel strategy for stem and progenitor cell therapy against cardiovascular ischemic diseases.

Cardiac progenitor cells (CPCs) are resident stem cells present in a small portion of ischemic hearts and function in repairing the damaged heart tissue. Intense oxidative stress impairs cell metabolism thereby decreasing cell viability. Protecting CPCs from undergoing cellular apoptosis during oxidative stress is crucial in optimizing CPC-based therapy. Histochrome (sodium salt of echinochrome A&mdash, a common sea urchin pigment) is an antioxidant drug that has been clinically used as a pharmacologic agent for ischemia/reperfusion injury in Russia. However, the mechanistic effect of histochrome on CPCs has never been reported. We investigated the protective effect of histochrome pretreatment on human CPCs (hCPCs) against hydrogen peroxide (H2O2)-induced oxidative stress. Annexin V/7-aminoactinomycin D (7-AAD) assay revealed that histochrome-treated CPCs showed significant protective effects against H2O2-induced cell death. The anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and Bcl-xL were significantly upregulated, whereas the pro-apoptotic proteins BCL2-associated X (Bax), H2O2-induced cleaved caspase-3, and the DNA damage marker, phosphorylated histone (&gamma, H2A.X) foci, were significantly downregulated upon histochrome treatment of hCPCs in vitro. Further, prolonged incubation with histochrome alleviated the replicative cellular senescence of hCPCs. In conclusion, we report the protective effect of histochrome against oxidative stress and present the use of a potent and bio-safe cell priming agent as a potential therapeutic strategy in patient-derived hCPCs to treat heart disease.

Cardiovascular diseases (CVDs) are a major cause of death worldwide. Due to the prevalence of many side effects and incomplete recovery from pharmacotherapies, stem cell therapy is being targeted for the treatment of CVDs. Among the different types of stem cells, endothelial progenitor cells (EPCs) have great potential. However, cellular replicative senescence decreases the proliferation, migration, and overall function of EPCs. Sirtuin 1 (SIRT1) has been mainly studied in the mammalian aging process. MHY2233 is a potent synthetic SIRT1 activator and a novel antiaging compound. We found that MHY2233 increased the expression of SIRT1, and its deacetylase activity thereby decreased expression of the cellular senescence biomarkers, p53, p16, and p21. In addition, MHY2233 decreased senescence-associated beta-galactosidase- (SA-β-gal-) positive cells and senescence-associated secretory phenotypes (SASPs), such as the secretion of interleukin- (IL-) 6, IL-8, IL-1α, and IL-1β. MHY2233 treatment protected senescent EPCs from oxidative stress by decreasing cellular reactive oxygen species (ROS) levels, thus enhancing cell survival and function. The angiogenesis, proliferation, and migration of senescent EPCs were enhanced by MHY2233 treatment. Thus, MHY2233 reduces replicative and oxidative stress-induced senescence in EPCs. Therefore, this novel antiaging compound MHY2233 might be considered a potent therapeutic agent for the treatment of age-associated CVDs.

There is a minor spelling error in the last of name of the 9th author in the originally published article.

Cardiovascular diseases (CVDs) are a major cause of death worldwide. Due to the prevalence of many side effects and incomplete recovery from pharmacotherapies, stem cell therapy is being targeted for the treatment of CVDs. Among the different types of stem cells, endothelial progenitor cells (EPCs) have great potential. However, cellular replicative senescence decreases the proliferation, migration, and overall function of EPCs. Sirtuin 1 (SIRT1) has been mainly studied in the mammalian aging process. MHY2233 is a potent synthetic SIRT1 activator and a novel antiaging compound. We found that MHY2233 increased the expression of SIRT1, and its deacetylase activity thereby decreased expression of the cellular senescence biomarkers, p53, p16, and p21. In addition, MHY2233 decreased senescence-associated beta-galactosidase- (SA-β-gal-) positive cells and senescence-associated secretory phenotypes (SASPs), such as the secretion of interleukin- (IL-) 6, IL-8, IL-1α, and IL-1β. MHY2233 treatment protected senescent EPCs from oxidative stress by decreasing cellular reactive oxygen species (ROS) levels, thus enhancing cell survival and function. The angiogenesis, proliferation, and migration of senescent EPCs were enhanced by MHY2233 treatment. Thus, MHY2233 reduces replicative and oxidative stress-induced senescence in EPCs. Therefore, this novel antiaging compound MHY2233 might be considered a potent therapeutic agent for the treatment of age-associated CVDs.

Endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs) play a pivotal role in vascular regeneration in ischemic tissues; however, their therapeutic application in clinical settings is limited due to the low quality and quantity of patient-derived circulating EPCs. To solve this problem, we evaluated whether three priming small molecules (tauroursodeoxycholic acid, fucoidan, and oleuropein) could enhance the angiogenic potential of EPCs. Such enhancement would promote the cellular bioactivities and help to develop functionally improved EPC therapeutics for ischemic diseases by accelerating the priming effect of the defined physiological molecules. We found that preconditioning of each of the three small molecules significantly induced the differentiation potential of CD34+ stem cells into EPC lineage cells. Notably, long-term priming of OECs with the three chemical cocktail (OEC-3C) increased the proliferation potential of EPCs via ERK activation. The migration, invasion, and tube-forming capacities were also significantly enhanced in OEC-3Cs compared with unprimed OECs. Further, the cell survival ratio was dramatically increased in OEC-3Cs against H2O2-induced oxidative stress via the augmented expression of Bcl-2, a prosurvival protein. In conclusion, we identified three small molecules for enhancing the bioactivities of ex vivo-expanded OECs for vascular repair. Long-term 3C priming might be a promising methodology for EPC-based therapy against ischemic diseases.

Cross talks between the renin-angiotensin system (RAS), sympathetic nervous system, and vascular homeostasis are tightly coordinated in hypertension. Angiotensin II (Ang II), a key factor in RAS, when abnormally activated, affects the number and bioactivity of circulating human endothelial progenitor cells (hEPCs) in hypertensive patients. In this study, we investigated how the augmentation of Ang II regulates adrenergic receptor-mediated signaling and angiogenic bioactivities of hEPCs. Interestingly, the short-term treatment of hEPCs with Ang II drastically attenuated the expression of beta-2 adrenergic receptor (ADRB2), but did not alter the expression of beta-1 adrenergic receptor (ADRB1) and Ang II type 1 receptor (AT1R). EPC functional assay clearly demonstrated that the treatment with ADRB2 agonists significantly increased EPC bioactivities including cell proliferation, migration, and tube formation abilities. However, EPC bioactivities were decreased dramatically when treated with Ang II. Importantly, the attenuation of EPC bioactivities by Ang II was restored by treatment with an AT1R antagonist (telmisartan; TERT). We found that AT1R binds to ADRB2 in physiological conditions, but this binding is significantly decreased in the presence of Ang II. Furthermore, TERT, an Ang II-AT1R interaction blocker, restored the interaction between AT1R and ADRB2, suggesting that Ang II might induce the dysfunction of EPCs via downregulation of ADRB2, and an AT1R blocker could prevent Ang II-mediated ADRB2 depletion in EPCs. Taken together, our report provides novel insights into potential therapeutic approaches for hypertension-related cardiovascular diseases.

Diabetic cardiomyopathy (DCM) is tightly linked to heart disorders and dysfunction or death of the cardiomyocytes including resident cardiac progenitor cells (CPCs) in diabetic patients. In order to restore loss of function of resident or transplanted CPCs, much research has focused on novel therapeutic strategies including the discovery of novel function-modulating factors such as reactive oxygen species (ROS) scavengers. Here, we developed and defined a novel antioxidant, MHY-1684, for enhancing the angiogenic potential of CPCs against ROS-related DCM. Short-term treatment with MHY-1684 restored ROS-induced CPC cell death. Importantly, MHY-1684 decreased hyperglycemia-induced mitochondrial ROS generation and attenuated hyperglycemia-induced mitochondrial fragmentation. We observed that the activation process of both Drp1 (phosphorylation at the site of Ser616) and Fis-1 is drastically attenuated when exposed to high concentrations of D-glucose with MHY-1684. Interestingly, phosphorylation of Drp1 at the site of Ser637, which is an inhibitory signal for mitochondrial fusion, is restored by MHY-1684 treatment, suggesting that this antioxidant may affect the activation and inhibition of mitochondrial dynamics-related signaling and mitochondrial function in response to ROS stress. In conclusion, our finding of the novel compound, MHY-1684, as an ROS scavenger, might provide an effective therapeutic strategy for CPC-based therapy against diabetic cardiomyopathy.

The purpose of the present study is to improve the endothelial progenitor cells (EPC) activation, proliferation, and angiogenesis using enzyme-aided extraction of fucoidan by amyloglucosidase (EAEF-AMG). Enzyme-aided extraction of fucoidan by AMG (EAEF-AMG) significantly increased EPC proliferation by reducing the reactive oxygen species (ROS) and decreasing apoptosis. Notably, EAEF-AMG treated EPCs repressed the colocalization of TSC2/LAMP1 and promoted perinuclear localization of mTOR/LAMP1 and mTOR/Rheb. Moreover, EAEF-AMG enhanced EPC functionalities, including tube formation, cell migration, and wound healing via regulation of AKT/Rheb signaling. Our data provided cell priming protocols to enhance therapeutic applications of EPCs using bioactive compounds for the treatment of CVD.

Doxorubicin (DOXO) is widely used to treat solid tumors. However, its clinical use is limited by side effects including serious cardiotoxicity due to cardiomyocyte damage. Resident cardiac progenitor cells (hCPCs) act as key regulators of homeostasis in myocardial cells. However, little is known about the function of hCPCs in DOXO-induced cardiotoxicity. In this study, we found that DOXO-mediated hCPC toxicity is closely related to calcium-related autophagy signaling and was significantly attenuated by blocking mTOR signaling in human hCPCs. DOXO induced hCPC apoptosis with reduction of SMP30 (regucalcin) and autophagosome marker LC3, as well as remarkable induction of the autophagy-related markers, Beclin-1, APG7, and P62/SQSTM1 and induction of calcium-related molecules, CaM (Calmodulin) and CaMKII (Calmodulin kinase II). The results of an LC3 puncta assay further indicated that DOXO reduced autophagosome formation via accumulation of cytosolic Ca2+. Additionally, DOXO significantly induced mTOR expression in hCPCs, and inhibition of mTOR signaling by rapamycin, a specific inhibitor, rescued DOXO-mediated autophagosome depletion in hCPCs with significant reduction of DOXO-mediated cytosolic Ca2+ accumulation in hCPCs, and restored SMP30 and mTOR expression. Thus, DOXO-mediated hCPC toxicity is linked to Ca2+-related autophagy signaling, and inhibition of mTOR signaling may provide a cardio-protective effect against DOXO-mediated hCPC toxicity.

Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes.

Endothelial progenitor cells (EPCs) are a promising cell source for the treatment of several ischemic diseases for their potentials in neovascularization. However, the application of EPCs in cell-based therapy has shown low therapeutic efficacy due to hostile tissue conditions after ischemia. In this study, a bio-blood-vessel (BBV) is developed, which is produced using a novel hybrid bioink (a mixture of vascular-tissue-derived decellularized extracellular matrix (VdECM) and alginate) and a versatile 3D coaxial cell printing method for delivering EPC and proangiogenic drugs (atorvastatin) to the ischemic injury sites. The hybrid bioink not only provides a favorable environment to promote the proliferation, differentiation, and neovascularization of EPCs but also enables a direct fabrication of tubular BBV. By controlling the printing parameters, the printing method allows to construct BBVs in desired dimensions, carrying both EPCs and atorvastatin-loaded poly(lactic-co-glycolic) acid microspheres. The therapeutic efficacy of cell/drug-laden BBVs is evaluated in an ischemia model at nude mouse hind limb, which exhibits enhanced survival and differentiation of EPCs, increased rate of neovascularization, and remarkable salvage of ischemic limbs. These outcomes suggest that the 3D-printed ECM-mediated cell/drug implantation can be a new therapeutic approach for the treatment of various ischemic diseases.