Yuebing Wang

Sonic hedgehog promotes autophagy of vascular smooth muscle cells

Sonic hedgehog (Shh) is a morphogen critically involved in development that is reexpressed in atherosclerotic lesions. It also stimulates proliferation of vascular smooth muscle cells (SMCs). Autophagy in vascular SMCs is known to promote SMC survival and increase plaque stability. The aim of this study was to investigate whether Shh induces autophagy of vascular SMCs. Our study showed that both Shh protein and microtubule-associated protein 1 light chain 3 (LC3)-II were increased in SMCs within neointimal lesions of mouse common carotid arteries. In cultured mouse aortic SMCs, recombinant mouse Shh stimulated LC3-II levels. Overexpression of wild-type mouse Shh through the tetracycline-regulated expression-inducible system in human aortic SMCs time-dependently increased the levels of LC3-II and also stimulated protein kinase B (AKT) phosphorylation. Pretreatment with AKT inhibitor IV (AKTI IV) inhibited AKT phosphorylation and the increase in LC3-II. Shh-induced autophagy was further confirmed by the formation of autophagosomes as detected by immunostaining and transmission electron microscopy, which was inhibited by AKTI IV. Shh further increased SMC LC3-II in the presence of bafilomycin A1, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, and pepstatin A or siRNA for the autophagy-related gene 7 (ATG7). In addition, Shh induced SMC proliferation, which was inhibited not only by AKTI IV but also by cyclopamine, an inhibitor of Shh receptor. Inhibition of autophagy with 3-methyladenine (3-MA), bafilomycin A1, or ATG7 siRNA resulted in inhibition of cell proliferation. Treatment with 3-MA, AKTI IV, or cyclopamine inhibited neointima formation in mouse common carotid arteries. Taken together, our results have shown that Shh induces autophagy of vascular SMCs involving AKT activation, suggesting a role of autophagy in Shh-induced cellular responses.

Molecular imaging for assessment of mesenchymal stem cells mediated breast cancer therapy

The tumor tropism of mesenchymal stem cells (MSCs) makes them an excellent delivery vehicle used in anticancer therapy. However, the exact mechanisms of MSCs involved in tumor microenvironment are still not well defined. Molecular imaging technologies with the versatility in monitoring the therapeutic effects, as well as basic molecular and cellular processes in real time, offer tangible options to better guide MSCs mediated cancer therapy. In this study, an in situ breast cancer model was developed with MDA-MB-231 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP). In mice breast cancer model, we injected human umbilical cord-derived MSCs (hUC-MSCs) armed with a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk), renilla luciferase (Rluc) and red fluorescent protein (RFP) into tumor on day 13, 18, 23 after MDA-MB-231 cells injection. Bioluminescence imaging of Fluc and Rluc provided the real time monitor of tumor cells and hUC-MSCs simultaneously. We found that tumors were significantly inhibited by hUC-MSCs administration, and this effect was enhanced by ganciclovir (GCV) application. To further demonstrate the effect of hUC-MSCs on tumor cells in vivo, we employed the near infrared (NIR) imaging and the results showed that hUC-MSCs could inhibit tumor angiogenesis and increased apoptosis to a certain degree. In conclusion, hUC-MSCs can inhibit breast cancer progression by inducing tumor cell death and suppressing angiogenesis. Moreover, molecular imaging is an invaluable tool in tracking cell delivery and tumor response to hUC-MSCs therapies as well as cellular and molecular processes in tumor.

Differential expression of G-protein-coupled estrogen receptor-30 in human myometrial and uterine leiomyoma smooth muscle

OBJECTIVE To determine differential expression of G-protein-coupled receptor 30 (GPR30) in uterine leiomyoma and its matched myometrium. DESIGN GPR30 expression examined in both tissues and cultured cells. SETTING Research laboratories. PATIENT(S) Women 35 to 50 years old with uterine leiomyomas. INTERVENTION(S) Hysterectomy. MAIN OUTCOME MEASURE(S) GPR30 expression profile. RESULT(S) Using Western blot and real-time quantitative polymerase chain reaction analyses, we found that GPR30 was highly expressed in uterine leiomyomas compared with their matched myometrium. In only three out of nine patients examined was GPR30 protein detectable by Western blot analysis in myometrial tissues, but at statistically significantly lower levels than in their leiomyomas. Confocal microscopy revealed the nuclear localization of GPR30 in leiomyoma tissues and cultured leiomyoma smooth muscle cells (SMCs). Treatment with 0.1 μM 17β-estradiol increased mRNA expression of GPR30 in leiomyoma SMCs but decreased expression in myometrial SMCs. Treatment with G-1, a GPR30 agonist, stimulated phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in both SMC types. PD98059, the MEK inhibitor, completely inhibited G-1-induced phosphorylation of p44/42 in myometrium SMCs, but not in SMCs from leiomyoma. CONCLUSION(S) GPR30 is abundantly expressed in uterine leiomyomas, likely resulting from estrogen stimulation.

Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment

The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes’ expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.

Nanoparticle-Based Tumor Theranostics with Molecular Imaging

The past few decades have brought on a dramatic change in the treatment of cancer; however, routine treatments fail to specifically clear tumorigenic cells and may be followed by cancer recurrence. Recent advances in developing multifunctional nanoparticles provide exciting new possibilities for drug delivery used in tumor-targeted therapy. Moreover, molecular imaging is an invaluable tool in evaluating new molecular targets, cancer diagnosis, predicting of tumor response to available therapies and monitoring response to therapy as well as developing new drugs prior to clinical translation. Combining of targeted cancer therapy and molecular imaging, termed as image-guided drug delivery, can achieve objectives of noninvasive assessment of drug biodistribution and real-time monitoring of therapeutic responses. Image-guided drug delivery has become an upcoming field with extensive prospect in cancer therapy and may provide an effective platform for personalized cancer care. This review will focus on the significance and recent advances in targeted and traceable therapeutic strategy for cancer therapy.

Increased expression of tuberin in human uterine leiomyoma

Female Eker rats harboring an insertional deletion in one copy of the tuberous sclerosis complex 2 (Tsc2) gene develop uterine leiomyoma, but the underlying mechanism of human uterine leiomyoma is not completely understood. To examine whether down-regulation of tuberin, a TSC2 gene product, is present in human uterine leiomyoma, we analyzed leiomyoma and matched myometrium tissues from 22 Chinese patients with Western blotting and real-time polymerase chain reaction analyses, and found that the expression of tuberin was significantly increased in leiomyoma tissues compared with matched myometrium tissues with inhibition of both the mammalian target of rapacmycin pathway and mitogen-activated protein kinase pathways.

Rapamycin inhibits hydrogen peroxide-induced loss of vascular contractility

Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, has been shown to extend the life span of mice, and oxidative stress plays critical roles in vascular aging involving loss of compliance of arteries. We examined, therefore, whether rapamycin has protective effects on the inhibition of vascular contractility by hydrogen peroxide (H₂O₂). Prolonged (3 h) exposure to H₂O₂ induced complete loss of contraction of mouse aortic rings and mesenteric (resistance) arteries to either KCl or phenylephrine, which was attenuated by pretreatment with rapamycin. H₂O₂-induced loss of contractility was unaffected by treatment with actinomycin D or cycloheximide, inhibitors of gene transcription and protein synthesis, respectively. Western blot analysis showed that there was no increase in phosphorylation of S6 kinase 1 (S6K) or factor 4E binding protein 1 (4EBP1) in response to H₂O₂ treatment, suggesting involvement of the mTOR complex-2 (mTORC2) rather than mTORC1. H₂O₂ treatment inhibited phosphorylation of the 20-kDa regulatory light chains of myosin (LC₂₀), which was partially blocked by rapamycin treatment. Interestingly, the calcineurin inhibitors cyclosporine A and FK506 were found to mimic the rapamycin effect, and rapamycin inhibited calcineurin activation induced by H₂O₂. We conclude that rapamycin inhibits H₂O₂-induced loss of vascular contractility, likely through an mTORC2-calcineurin pathway.

Atorvastatin Inhibits Myocardin Expression in Vascular Smooth Muscle Cells

Atorvastatin (ATV), an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is widely prescribed as a lipid-lowering drug. It also inhibits the RhoA-Rho–associated kinase pathway in vascular smooth muscle (SM) cells and critically inhibits SM function. Myocardin is a coactivator of serum response factor, which upregulates SM contractile proteins. The RhoA-Rho–associated kinase pathway, which directly triggers SM contraction, also increases myocardin gene expression. Therefore, we investigated whether ATV inhibits myocardin gene expression in SM cells. In mice injected with ATV (IP 20 &mgr;g/g per day) for 5 days, myocardin gene expression was significantly downregulated in aortic and carotid arterial tissues with decreased expression of myocardin target genes SM &agr;-actin and SM22. Correspondingly, the contractility of aortic rings in mice treated with ATV or the Rho–associated kinase inhibitor Y-27632 was reduced in response to treatment with either KCl or phenylephrine. In cultured mouse and human aortic SM cells, KCl treatment stimulated the expression of myocardin, SM &agr;-actin, and SM22. These stimulatory effects were prevented by ATV treatment. ATV-induced inhibition of myocardin expression was prevented by pretreatment with either mevalonate or geranylgeranylpyrophosphate but not farnesylpyrophosphate. Treatment with Y-27632 mimicked ATV effects on the gene expression of myocardin, SM &agr;-actin, and SM22, further suggesting a role for the RhoA-Rho–associated kinase pathway in ATV effects. Furthermore, ATV treatment inhibited RhoA membrane translocation and activation; these effects were prevented by pretreatment with mevalonate. We conclude that ATV inhibits myocardin gene expression in vivo and in vitro, suggesting a novel mechanism for ATV inhibition of vascular contraction.

Embryonic stem cell preconditioned microenvironment suppresses tumorigenic properties in breast cancer

BackgroundMicroenvironment is being increasingly recognized as a critical determinant in tumor progression and metastasis. However, the appropriate regulatory mechanism to maintain the normal balance between differentiation and self-renewal of the cancer cell in microenvironment is not well known.Methods4T1 breast cancer cells were treated with embryonic stem (ES) cell conditioned medium which was collected from mouse ES cells. Inhibition of tumor cell growth was based on the reduction of cell proliferation and viability, and inhibition of aggressive properties of tumor cells were examined using the wound-healing and mammosphere assays. The expression of stem cell-associated genes was detected by quantitative RT-PCR.ResultsWe used a real-time imaging system to investigate the effect of the mouse ES cell microenvironment on aggressive breast cancer cells in vitro and in vivo. Exposure of breast cancer cells in mouse ES cell conditioned medium resulted in inhibition of growth, migration, metastasis, and angiogenesis of cancer cells. For many tumors, aggressive properties were tightly related to Stat3 signaling activation. We specifically discovered that the ES cell microenvironment sufficiently suppressed Stat3 signaling pathway activation in aggressive tumor cells, leading to a reduction in tumorigenesis and invasiveness.ConclusionsWe identified important functions of Stat3 and their implications for antitumor effects of ES cell conditioned medium. Some factors secreted by ES cells could efficiently suppress Stat3 pathway activation in breast cancer cells, and were then involved in cancer cell growth, survival, invasion, and migration. This study may act as a platform to understand tumor cell plasticity and may offer new therapeutic strategies to inhibit breast cancer progression.

The Phenotypic Fate of Bone Marrow-Derived Stem Cells in Acute Kidney Injury

Background: Despite increasing attention on the role of bone marrow derived stem cells in repair or rejuvenation of tissues and organs, cellular mechanisms of such cell-based therapy remain poorly understood. Methods: We reconstituted hematopoiesis in recipient C57BL/6J mice by transplanting syngeneic GFP+ bone marrow (BM) cells. Subsequently, the recipients received subcutaneous injection of granulocyte-colony stimulating factor (G-CSF) and were subjected to acute renal ischemic injury. Flow cytometry and immunostaining were performed at various time points to assess engraftment and phenotype of BM derived stem cells. Results: Administration of G-CSF increased the release of BM derived stem cells into circulation and enhanced the ensuing recruitment of BM derived stem cells into injured kidney. During the second month post injury, migrated BM derived stem cells lost hematopoietic phenotype (CD45) but maintained the expression of other markers (Sca-1, CD133 and CD44), suggesting their potential of transdifferentiation into renal stem cells. Moreover, G-CSF treatment enhanced the phenotypic conversion. Conclusion: Our work depicted a time-course dependent transition of phenotypic characteristics of BM derived stem cells, demonstrated the existence of BM derived stem cells in damaged kidney and revealed the effects of G-CSF on cell transdifferentiation.