Hong-Bo Xin

RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

Bone Marrow–Derived Smooth Muscle–Like Cells Are Infrequent in Advanced Primary Atherosclerotic Plaques but Promote Atherosclerosis

Objective—Although vascular smooth muscle cells (VSMCs) provide the major structural integrity of atherosclerotic plaques, their origin has been questioned. In particular, although some studies identified plaque VSMCs originating from bone marrow or peripheral blood, their frequency is controversial and their function unknown. We used genetic tracking of cell fate through smooth muscle cell (SMC)–specific LacZ reporter activity and VSMC-selective apoptosis to investigate the frequency, distribution, and role of marrow-derived VSMCs in atherogenesis. Methods and Results—Cultured mouse bone marrow–derived smooth muscle–like cells expressed SMC markers and functional SMC promoter-driven transgenes over time. Transplantation of apolipoprotein E (ApoE)−/− mice with smooth muscle myosin heavy chain–Cre/ROSA26R/ApoE−/− marrow showed that 0.7±0.14% cells expressed LacZ in atherosclerotic plaques, located superficially in early plaques, and in necrotic cores but not fibrous caps of advanced lesions. Cells expressing both progenitor and SMC markers showed a similar distribution and frequency. Apoptosis of marrow-derived SMC-like cells transplanted from SM22&agr;–human diphtheria toxin receptor/ApoE−/− mice retarded atherogenesis, with reduced plaque macrophage content. Cultured marrow-derived SMC-like cells secreted proinflammatory cytokines and promoted macrophage migration, VSMC proliferation, and collagen synthesis. Conclusion—Bone marrow–derived SMC-like cells are infrequent in advanced primary atherosclerotic plaques and absent in fibrous caps. However, these cells secrete proinflammatory cytokines and mitogens and promote atherosclerosis.

Involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells

Glioblastoma is one of the most aggressive brain tumors with high morbidity and mortality. Hypoxia is often the common characteristic of tumor microenvironment, and hypoxia-inducible factor-1α (HIF-1α) is an essential factor regulating the migratory activity of cancer cells including glioblastoma. Recently, mitochondrial dynamics was found to be involved in the aggression of cancer cells. However, whether dynamin-related protein 1 (Drp1) contributes to the migration of human glioblastoma cells under hypoxia remains unknown. In the present study, hypoxia was found to upregulate the transcription and expression of Drp1, and stimulated mitochondrial fission in glioblastoma U251 cells. Inhibition of HIF-1α with echinomycin blocked hypoxia‑induced expression of Drp1. Notably, Drp1 inhibitor Mdivi-1 efficiently attenuated hypoxia-induced mitochondrial fission and migration of U251 cells. In addition, three U251 stable cell lines expressing GFP, GFP-Drp1 and dominant negative GFP-Drp1‑K38A were established to examine the direct role of Drp1 in hypoxia-induced migration. MTT assay showed that there was no significant difference in proliferation of three cell lines. Compared with the GFP cell line, exogenously expressed GFP-Drp1-K38A inhibited hypoxia-induced migration of U251 cells, while stable expression of GFP-Drp1 enhanced the migration of U251 cells under hypoxia. Therefore, this study indicates the involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells, and suggests Drp1 to be a potential therapeutic target to suppress the aggression of glioblastoma in the future.

c-Myb–Dependent Smooth Muscle Cell Differentiation

Both in vitro and in vivo studies have implicated the c-Myb transcription factor in vascular smooth muscle cell (SMC) proliferation and hematopoiesis. However, its role in differentiation and maturation of contractile, as opposed to proliferating, SMCs has not been investigated. Here we demonstrate that c-myb−/− embryonic stem cells (ESCs) are incapable of producing embryoid bodies (EBs) with spontaneously contracting SMCs but can differentiate into contracting cardiomyocytes unimpaired. Quantitative real-time RT-PCR revealed that whereas mesodermal differentiation was unaffected, myocardin, a critical determinant of SMC differentiation, became upregulated at day 7 in wild-type, but not in c-myb−/− EBs. SMC-specific genes, smooth muscle &agr;-actin, SM22&agr; and smooth muscle myosin heavy chain reached peak expression levels by day 15 of differentiation and were 2- to 3-fold higher in wild-type as compared with c-myb−/− derived EBs. Similarly, fluorescence-activated cell-sorting analysis confirmed significantly different proportions of smooth muscle &agr;-actin–positive cells in wild-type (26.8±0.7%) versus c-myb−/− (12.3±0.4%) EBs. Temporal induction of these SMC-specific markers preceded and paralleled contractile SMC appearance and predicted the relative (in)ability of c-myb−/− and wild-type ESC lines to generate EBs with contracting SMCs. Importantly, data from EBs faithfully predicted a significant reduction in c-myb−/− cell contribution to SMC lineage in vivo, in chimeric E11.5 embryo and adult aortas relative to brain and skin chimerism, respectively. Moreover, the visceral SMC population in chimeric embryos was nearly devoid of c-myb−/− cells. Our data are the first to implicate c-Myb in SMC differentiation from precursor stem cell–derived populations, reinforcing its potential role in phenotypic modulation of SMCs and vascular disease.

Mitochondrial dynamics regulates hypoxia-induced migration and antineoplastic activity of cisplatin in breast cancer cells

Mitochondria are high dynamic organelles with frequent fission and fusion. Here, we found hypoxia stimulated Drp1 expression, mitochondrial fission and migration in metastatic MDA-MB‑231 cells, but not in non-metastatic MCF-7 cells. Inhibition of Drp1-dependent mitochondrial fission by Mdivi-1 or silencing Drp1 attenuated hypoxia-induced mitochondrial fission and migration in MDA-MB‑231 cells. On the other hand, cisplatin induced significant apoptosis and mitochondrial fission in MDA-MB‑231 cells, but not in MCF-7 cells. Mdivi-1 and silencing Drp1 also efficiently prevented cisplatin-induced MMP decrease, ROS production and apoptosis in MDA-MB‑231 cells. Our data suggest that Drp1-dependent mitochondrial fission not only regulates hypoxia-induced migration of breast cancer cells, but also facilitates its sensitivity to chemotherapeutic agents. Thus, targeting Drp1-dependent mitochondrial dynamics may provide a novel strategy to suppress breast cancer metastasis and improve the chemotherapeutic effect in the future.

Gene trap and gene inversion methods for conditional gene inactivation in the mouse

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.

CD38 Deficiency Protects the Heart from Ischemia/Reperfusion Injury through Activating SIRT1/FOXOs-Mediated Antioxidative Stress Pathway

Ischemia/reperfusion (I/R) injury induces irreversible oxidative stress damage to the cardiac muscle. We previously observed that CD38 deficiency remarkably protects mouse embryonic fibroblasts (MEFs) from oxidative stress-induced injury. However, whether CD38 deficiency protects from I/R injury in the heart is not explored. Here, we showed that the hearts of CD38 deficient mice or wild type mice supplied with exogenous NAD were significantly protected from ischemia/reperfusion injury, seen as reduction of the myocardial infarct sizes when the mice were subjected to 30 min ischemia followed by 24 hours of reperfusion. Consistently, the protection of CD38 deficiency on hypoxia/reoxygenation (H/R) injury was confirmed with a CD38 knockdown H9c2 stable cell line. Furthermore, we observed that knockdown of CD38 remarkably inhibited ROS generation and intracellular Ca2+ overloading induced by H/R in H9c2 cells. The FOXO1 and FOXO3 expressions were significantly elevated by H/R injury in CD38 knockdown cells compared with normal H9c2 cells. The cell immunofluorescence assay showed that FOXO1 nuclear translocation was significantly increased in CD38 knockdown H9c2 cells. In addition, we demonstrated that the increase of FOXO1 nuclear translocation was associated with the increased expressions of antioxidant catalase and SOD2 and the attenuated expression of the ROS generation enzyme NOX4. In conclusion, our results provide new evidence that CD38 deficiency protects the heart from I/R injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway.

Asiatic Acid Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β/HIF-1α Signaling in Rat H9c2 Cardiomyocytes

Myocardial ischemic/reperfusion injury results from severe impairment of coronary blood supply and leads to irreversible cell death, with limited therapeutic possibilities. Asiatic acid is a pentacyclic triterpenoid derived from the tropical medicinal plant Centella asiatica and serves a variety of bioactivities. In this study, we determined the effect of asiatic acid on myocardial ischemia/reperfusion injury and investigated the underlying mechanisms, using an in vitro rat H9c2 cardiomyocytes model of oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Results showed that pre-treatment with asiatic acid significantly augmented cell viability and prevented lactate dehydrogenase (LDH) release in a concentration-dependent manner after OGD/R exposure. Asiatic acid at 10 μM effectively inhibited apoptotic cell death, suppressed the activities of caspase-3 and caspase-9, and reversed Bax/Bcl-2 ratio in hypoxic H9c2 cells. In addition, asiatic acid improved mitochondrial function, as evidenced by reduced reactive oxygen species (ROS) accumulation, enhanced mitochondrial membrane potential and decreased intracellular calcium concentration. Using Western blot assay, we found that asiatic acid promoted the phosphorylation of Akt and subsequent inactivation of glycogen synthase kinase-3β (GSK-3β), and induced the expression of hypoxia-inducible factor 1α (HIF-1α) after OGD/R. The cardioprotective effects of asiatic acid were attenuated by the Akt or HIF-1α inhibitor. Taken together, these data suggested that asiatic acid exerted protective effects against OGD/R-induced apoptosis in cardiomyocytes, at least partly via the Akt/GSK-3β/HIF-1α pathway.

Zn or O? An Atomic Level Comparison on Antibacterial Activities of Zinc Oxides.

For the first time, the influence of different types of atoms (Zn and O) on the antibacterial activities of nanosized ZnO was quantitatively evaluated with the aid of a 3D-printing-manufactured evaluation system. Two different outermost atomic layers were manufactured separately by using an ALD (atomic layer deposition) method. Interestingly, we found that each outermost atomic layer exhibited certain differences against gram-positive or gram-negative bacterial species. Zinc atoms as outermost layer (ZnO-Zn) showed a more pronounced antibacterial effect towards gram-negative E. coli (Escherichia coli), whereas oxygen atoms (ZnO-O) showed a stronger antibacterial activity against gram-positive S. aureus (Staphylococcus aureus). A possible antibacterial mechanism has been comprehensively discussed from different perspectives, including Zn(2+) concentrations, oxygen vacancies, photocatalytic activities and the DNA structural characteristics of different bacterial species.

Post-transcriptional gene regulation by RNA-binding proteins in vascular endothelial dysfunction.

Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative and migratory capacity of endothelial cells, as well as control of leukocyte trafficking. Endothelial dysfunction is an early step in vascular inflammatory diseases such as atherosclerosis, diabetic vascular complications, sepsis-induced or severe virus infection-induced organ injuries. The expressions of inflammatory cytokines and vascular adhesion molecules induced by various stimuli, such as modified lipids, smoking, advanced glycation end products and bacteria toxin, significantly contribute to the development of endothelial dysfunction. The transcriptional regulation of inflammatory cytokines and vascular adhesion molecules has been well-studied. However, the regulation of those gene expressions at post-transcriptional level is emerging. RNA-binding proteins have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level in microRNA-dependent or independent manners. This review summarizes the latest insights into the roles of RNA-binding proteins in controlling vascular endothelial cell functions and their contribution to the pathogenesis of vascular inflammatory diseases.