Showing papers by "Lei Wei published in 2014"

Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreERT2; Ihhfl/fl mice.

Abstract: Previous observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency. To evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreER T2 ; Ihhfl/fl). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates. Only mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh. These findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.

Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

Abstract: To investigate if decreased histone deacetylase 4 (HDAC4) is associated with human osteoarthritis (OA) cartilage degeneration by releasing HDAC4 inhibition of runt-related transcription factor-2 (Runx2) resulting in increase of OA cartilage degeneration-related genes. The mRNA and protein levels of HDAC4, Runx2, matrix metalloproteinase (MMP)-13, Indian hedgehog (Ihh) and type X collagen were detected by performing real-time PCR (RT-PCR), western blotting and immunohistochemistry on specimens from human OA and normal cartilage. To further explore the mechanism of regulation of Runx2 and OA-related genes by HDAC4, changes in these OA-related genes were further quantified by RT-PCR after overexpression of HDAC4 and knockdown of HDAC4 by siRNA. Runx2 and MMP-13 promoter activities were measured by dual luciferase assays. The levels of HDAC4 in the cartilage from OA patients and healthy 40- to 60-year-old donors were decreased to 31% and 65% compared with specimens from 20- to 40-year-old healthy donors, respectively (P <0.05). Decreased HDAC4 was associated with increased Runx2 and other OA-related genes in human OA cartilage, specifically: MMP-13, Ihh and type X collagen. Exogenous HDAC4 decreased the mRNA levels of Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, ADAMTS-4 and -5, and increased the mRNA of type II collagen. In addition, the data also shows that overexpression of HDAC4 not only decreased the expression of interleukin (IL)-1β, Cox2 and iNos and increased the expression of aggrecan, but also partially blocked the effect of IL-1β on expression of catabolic events in human OA chondrocytes. HDAC4 also inhibited Runx2 promoter activity and MMP13 promotor activity in a dose-dependent manner. In contrast, inhibition of HDAC4 by TSA drug had an opposite effect. Our study is the first to demonstrate that decreased HDAC4 contributes, at least in part, to the pathogenesis of OA cartilage degeneration. Thus, HDAC4 may have chondroprotective properties by inhibiting Runx2 and OA-related genes.

Identification of α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis.

Abstract: Objective To determine if supplemental intra-articular alpha-2 macroglobulin (A2M) has a chondroprotective effect in a rat OA model.

ROCK1 isoform-specific deletion reveals a role for diet-induced insulin resistance

Abstract: Rho kinase (ROCK) isoforms regulate insulin signaling and glucose metabolism negatively or positively in cultured cell lines and skeletal muscle. However, the in vivo function of the ROCK1 isoform ...

Specific inhibition of HDAC4 in cardiac progenitor cells enhances myocardial repairs.

Abstract: We have recently shown that in vivo inhibition of histone deacetylase (HDAC) stimulates endogenous myocardial regeneration in infarcted hearts (Zhang L et al. J Pharmacol Exp Ther 341: 285–293, 2012). Furthermore, our observation demonstrates that HDAC inhibition promotes cardiogenesis, which is associated with HDAC4 reduction. However, it remains unknown as to whether specific inhibition of HDAC4 modulates cardiac stem cells (CSCs) to facilitate myocardial repair and to preserve cardiac performance. c-kit+ CSCs were isolated from adult mouse hearts and were transfected with HDAC4 siRNA to knockdown HDAC4 of c-kit+ CSCs. The transfection of HDAC4 siRNA caused a marked reduction of HDAC4 mRNA and proteins in c-kit+ CSCs. Mouse myocardial infarction (MI) was created to assess the effect of HDAC4 inhibition in c-kit+ CSCs on myocardial regeneration in vivo when cells were introduced into MI hearts. Transplantation of HDAC4 siRNA-treated c-kit+ CSCs into MI hearts improved ventricular function, attenuated ventricular remodeling, and promoted CSC-derived regeneration and neovascularization. Furthermore, Ki67 and BrdU positively proliferative myocytes increased in MI hearts receiving HDAC4 siRNA-treated c-kit+ CSCs compared with MI hearts engrafted with control siRNA-treated c-kit+ CSCs. In addition, compared with MI hearts engrafted with control adenoviral GFP-infected c-kit+ CSCs, MI hearts receiving adenoviral HDAC4-infected c-kit+ CSCs exhibited attenuated cardiac functional recovery, CSC-derived regeneration, and neovascularization, which was accompanied with adverse ventricular remodeling and decrease in Ki67 and BrdU positively proliferative myocytes. HDAC4 inhibition facilitated c-kit+ CSCs into the differentiation into cardiac lineage commitments in vitro, while HDAC4 overexpression attenuated c-kit+ CSC-derived cardiogenesis. Our results indicate that HDAC4 inhibition promotes CSC-derived cardiac regeneration and improves the restoration of cardiac function.

Demineralized bone matrix combined bone marrow mesenchymal stem cells, bone morphogenetic protein-2 and transforming growth factor-β3 gene promoted pig cartilage defect repair.

Abstract: Objectives To investigate whether a combination of demineralized bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) infected with adenovirus-mediated- bone morphogenetic protein (Ad-BMP-2) and transforming growth factor-β3 (Ad-TGF-β3) promotes the repair of the full-thickness cartilage lesions in pig model. Methods BMSCs isolated from pig were cultured and infected with Ad-BMP-2(B group), Ad-TGF-β3 (T group), Ad-BMP-2 + Ad-TGF-β3(BT group), cells infected with empty Ad served as a negative group(N group), the expression of the BMP-2 and TGF-β3 were confirmed by immunofluorescence, PCR, and ELISA, the expression of SOX-9, type II collagen(COL-2A), aggrecan (ACAN) in each group were evaluated by real-time PCR at 1w, 2w, 3w, respectively. The chondrogenic differentiation of BMSCs was evaluated by type II collagen at 21d with immunohistochemical staining. The third-passage BMSCs infected with Ad-BMP-2 and Ad-TGF-β3 were suspended and cultured with DBM for 6 days to construct a new type of tissue engineering scaffold to repair full-thickness cartilage lesions in the femur condyles of pig knee, the regenerated tissue was evaluated at 1,2 and 3 months after surgery by gross appearance, H&E, safranin O staining and O'driscoll score. Results Ad-BMP-2 and Ad-TGF-β3 (BT group) infected cells acquired strong type II collagen staining compared with Ad-BMP-2 (B group) and Ad-TGF-β3 (T group) along. The Ad-BMP-2 and Ad-TGF-β3 infected BMSCs adhered and propagated well in DBM and the new type of tissue engineering scaffold produced hyaline cartilage morphology containing a stronger type II collagen and safranin O staining, the O'driscoll score was higher than other groups. Conclusions The DBM compound with Ad-BMP-2 and Ad-TGF-β3 infected BMSCs scaffold has a good biocompatibility and could well induce cartilage regeneration to repair the defects of joint cartilage. This technology may be efficiently employed for cartilage lesions repair in vivo.

Indian hedgehog in synovial fluid is a novel marker for early cartilage lesions in human knee joint.

Abstract: To determine whether there is a correlation between the concentration of Indian hedgehog (Ihh) in synovial fluid (SF) and the severity of cartilage damage in the human knee joints, the knee cartilages from patients were classified using the Outer-bridge scoring system and graded using the Modified Mankin score. Expression of Ihh in cartilage and SF samples were analyzed with immunohistochemistry (IHC), western blot, and enzyme-linked immunosorbent assay (ELISA). Furthermore, we detected and compared Ihh protein levels in rat and mice cartilages between normal control and surgery-induced osteoarthritis (OA) group by IHC and fluorescence molecular tomography in vivo respectively. Ihh expression was increased 5.2-fold in OA cartilage, 3.1-fold in relative normal OA cartilage, and 1.71-fold in OA SF compared to normal control samples. The concentrations of Ihh in cartilage and SF samples was significantly increased in early-stage OA samples when compared to normal samples (r = 0.556; p < 0.001); however, there were no significant differences between normal samples and late-stage OA samples. Up-regulation of Ihh protein was also an early event in the surgery-induced OA models. Increased Ihh is associated with the severity of OA cartilage damage. Elevated Ihh content in human knee joint synovial fluid correlates with early cartilage lesions.

Indian Hedgehog, a critical modulator in osteoarthritis, could be a potential therapeutic target for attenuating cartilage degeneration disease

Abstract: The Hedgehog (Hh) family of proteins consists of Indian hedgehog (Ihh), sonic hedgehog (Shh), and desert hedgehog (Dhh). These proteins serve as essential regulators in a variety of developmental events. Ihh is mainly produced and secreted by prehypertrophic chondrocytes and regulates chondrocyte hypertrophy and endochondral bone formation during growth plate development. Tissue-specific deletion of the Ihh gene (targeted by Col2a1-Cre) causes early lethality in mice. Transgenic mice with induced Ihh expression exhibit increased chondrocyte hypertrophy and cartilage damage resembling human osteoarthritis (OA). During OA development, chondrocytes recapitulate the differentiation process that happens during the fetal status and which does not occur to an appreciable degree in adult articular cartilage. Ihh expression is up-regulated in human OA cartilage, and this upregulation correlates with OA progression and changes in chondrocyte morphology. A genetic study in mice further showed that conditional deletion of Ihh in chondrocytes attenuates OA progression, suggesting the possibility that blocking Ihh signaling can be used as a therapeutic approach to prevent or delay cartilage degeneration. However, Ihh gene deletion is currently not a therapeutic option as it is lethal in animals. RNA interference (RNAi) provides a means to knockdown Ihh without the severe side effects caused by chemical inhibitors. The currently available delivery methods for RNAi are nanoparticles and liposomes. Both have problems that need to be addressed. In the future, it will be necessary to develop a safe and effective RNAi delivery system to target Ihh signaling for preventing and treating OA.

MicroRNA-1 regulates chondrocyte phenotype by repressing histone deacetylase 4 during growth plate development

Abstract: MicroRNAs (miRs) are noncoding RNAs (17–25 nt) that control translation and/or mRNA degradation. Using Northern blot analysis, we identified that miR-1 is specifically expressed in growth plate cartilage in addition to muscle tissue, but not in brain, intestine, liver, or lung. We obtained the first evidence that miR-1 is highly expressed in the hypertrophic zone of the growth plate, with an 8-fold increase compared with the proliferation zone; this location coincides with the Ihh and Col X expression regions in vivo. MiR-1 significantly induces chondrocyte proliferation and differentiation. We further identified histone deacetylase 4 (HDAC4) as a target of miR-1. HDAC4 negatively regulates chondrocyte hypertrophy by inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. MiR-1 inhibits both endogenous HDAC4 protein by 2.2-fold and the activity of a reporter gene bearing the 3′-untranslated region (UTR) of HDAC4 by 3.3-fold. Conversely, knockdown of endogenous miR-1 relieves the repression of HDAC4. Deletion of the miR-1 binding site in HDAC4 3′-UTR or mutated miR-1 abolishes miR-1-mediated inhibition of the reporter gene activity. Overexpression of HDAC4 reverses miR-1 induction of chondrocyte differentiation markers Col X and Ihh. HDAC4 inhibits Runx2 promoter activity in a dosage-dependent manner. Thus, miR-1 plays an important role in the regulation of the chondrocyte phenotype during the growth plate development via direct targeting of HDAC4. — Li, P., Wei, X., Guan, Y., Chen, Q., Zhao, T., Sun, C., Wei, L. MicroRNA-1 regulates chondrocyte phenotype by repressing histone deacetylase 4 during growth plate development.

Expansion on a matrix deposited by nonchondrogenic urine stem cells strengthens the chondrogenic capacity of repeated-passage bone marrow stromal cells

Abstract: Human urine-derived stem cells (hUSCs) are a newly found type of stem cell with a potential for therapeutic application in urology. The aim of this study is to investigate whether hUSCs contribute to cartilage regeneration. Despite their characterization with multi-lineage differentiation capacities, in terms of osteogenesis, adipogenesis and myogenesis, hUSCs do not show the ability to differentiate into chondrocytes. Human bone marrow stromal cells (hBMSCs) are a tissue-specific stem cell for endochondral bone formation; however, repeated-passage hBMSCs have a lower capacity for chondrogenic differentiation. We found that the extracellular matrix (ECM) deposited by hUSCs (UECM) can greatly recharge repeated-passage hBMSCs toward chondrogenic differentiation, a result that might be explained by trophic factors released from hUSCs being immobilized in UECM. We also found that ECM from repeated-passage hBMSCs (BECM) have a limited rejuvenation effect. The Wnt11-mediated noncanonical signaling pathway might be responsible for UECM-mediated hBMSC rejuvenation and subsequent chondrogenic differentiation. Our data indicate that commercially available UECM from young healthy donors might represent a simple and promising approach for autologous hBMSC rejuvenation. This study also provides an excellent model for investigating the effect of trophic factors released by stem cells on tissue regeneration without interference by stem cell differentiation.

TNF-α Mediated Increase of HIF-1α Inhibits VASP Expression, Which Reduces Alveolar-Capillary Barrier Function during Acute Lung Injury (ALI)

Abstract: Acute lung injury (ALI) is an inflammatory disorder associated with reduced alveolar-capillary barrier function and increased pulmonary vascular permeability. Vasodilator-stimulated phosphoprotein (VASP) is widely associated with all types of modulations of cytoskeleton rearrangement-dependent cellular morphology and function, such as adhesion, shrinkage, and permeability. The present studies were conducted to investigate the effects and mechanisms by which tumor necrosis factor-alpha (TNF-α) increases the tight junction permeability in lung tissue associated with acute lung inflammation. After incubating A549 cells for 24 hours with different concentrations (0–100 ng/mL) of TNF-α, 0.1 to 8 ng/mL TNF-α exhibited no significant effect on cell viability compared with the 0 ng/mL TNF-α group (control group). However, 10 ng/mL and 100 ng/mL TNF-α dramatically inhibited the viability of A549 cells compared with the control group (*p<0.05). Monolayer cell permeability assay results indicated that A549 cells incubated with 10 ng/mL TNF-α for 24 hours displayed significantly increased cell permeability (*p<0.05). Moreover, the inhibition of VASP expression increased the cell permeability (*p<0.05). Pretreating A549 cells with cobalt chloride (to mimic a hypoxia environment) increased protein expression level of hypoxia inducible factor-1α (HIF-1α) (*p<0.05), whereas protein expression level of VASP decreased significantly (*p<0.05). In LPS-induced ALI mice, the concentrations of TNF-α in lung tissues and serum significantly increased at one hour, and the value reached a peak at four hours. Moreover, the Evans Blue absorption value of the mouse lung tissues reached a peak at four hours. The HIF-1α protein expression level in mouse lung tissues increased significantly at four hours and eight hours (**p<0.001), whereas the VASP protein expression level decreased significantly (**p<0.01). Taken together, our data demonstrate that HIF-1α acts downstream of TNF-α to inhibit VASP expression and to modulate the acute pulmonary inflammation process, and these molecules play an important role in the impairment of the alveolar-capillary barrier.

Direct Renin Inhibition With Aliskiren Protects Against Myocardial Ischemia/Reperfusion Injury by Activating Nitric Oxide Synthase Signaling in Spontaneously Hypertensive Rats

Abstract: Background We tested the hypothesis that direct renin inhibition with aliskiren protects against myocardial ischemia/reperfusion (I/R) injury in spontaneously hypertensive rats (SHR), and examined the mechanism by which this occurs. Methods and Results Male SHR were treated (orally, 4 weeks) with saline or aliskiren (30 or 60 mg kg−1 day−1) and subjected to 30 minutes of left anterior descending coronary artery occlusion followed by 6 or 24 hours of reperfusion. Only the higher dose significantly lowered systolic blood pressure, the lower dose causing a smaller apparent lowering that was nonsignificant. Despite this difference in blood pressure-lowering effect, both doses increased the ejection fraction and fractional shortening and reduced myocardial infarct size equally. I/R decreased cardiac expression of phosphatidylinositol 3-kinase (PI3K), phospho-Akt and phospho-endothelial nitric oxide synthase (phospho-eNOS), but increased expression of inducible nitric oxide synthase (iNOS); these changes were all abrogated by aliskiren. Moreover, aliskiren decreased superoxide anion generation and increased cyclic guanosine-3′,5′-monophosphate, an index of bioactive nitric oxide, in myocardium. It also decreased the expression of myocardial matrix metalloproteinase-2, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 (TIMP-1) following I/R. In a Langendorff heart preparation, the detrimental cardiac effects of I/R were abrogated by aliskiren, and these protective effects were abolished by NOS or PI3K inhibition. In a parallel study, although specific iNOS inhibition reduced plasma malondialdehyde and myocardial superoxide anion generation, it did not affect the deleterious effects of I/R on myocardial structure and function. Conclusions Direct renin inhibition protects against myocardial I/R injury through activation of the PI3K-Akt-eNOS pathway.

Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure.

Abstract: Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure. The pathological significance of Rnd3 loss in the transition to heart failure remains unexplored. To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism, we generated Rnd3+/− haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3+/− mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3+/− haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by fasudil treatment partially improved Rnd3+/− mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho-associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double-knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3+/−/ROCK1−/−). Again, genetic deletion of ROCK1 partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype. These data suggest that downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients. Animals with Rnd3 haploinsufficiency are predisposed to hemodynamic stress. Hyperactivation of Rho kinase activity is responsible in part for the apoptotic cardiomyopathy development. Further investigation of ROCK1-independent mechanisms in Rnd3-mediated cardiac remodeling should be the focus for future study.

ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment.

Abstract: We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1−/− and ROCK2−/− MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1−/− MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.

The Protective Effect of Curcumin on Hepatotoxicity and Ultrastructural Damage Induced by Cisplatin

Abstract: We investigate the protective effect of curcumin (CU) on the hepatic ultrastructural damage induced by cisplatin in mice. 18 adult Kunming mice were randomly divided into normal saline (NS) group, cisplatin treatment group (CP) and CU + CP group (n = 6 for each group). Mice in control group and CP group were administered with NS (20 mL/kg/day) and CU + CP group were i.p injected with CU (200 mg/kg/day) for 10 days. Then cisplatin (50 mg/kg/day) was injected in mice of CP group and CU + CP group, while those in control group were given the same volume of NS. Five days after injection all mice were killed and liver dissected. The hepatic morphological structures were observed under light microscope and transmission electron microscope. The results indicated that CU alleviated the hepatic histopathological damages induced by cisplatin, which included declined body weight, vacuolated cytoplasm and blurred liver trabecular structure. Moreover, no hepatic ultrastructural damages were observed in the CU ...

Ritonavir-Induced Hepatotoxicity and Ultrastructural Changes of Hepatocytes

Abstract: To investigate the effect of ritonavir on hepatocyte proliferation, we detected the change of cleaved caspase-3 expression level in the hepatocytes. Furthermore, the morphological and ultrastructural changes of hepatocytes derived from RTV-treated mice have been observed. The results showed that ritonavir can evidently inhibit hepatocyte proliferation and increase cleaved caspase-3 expression level. Under the electron microscope, chromatin margination, mitochondrial cristae disappearance, karyopyknosis and cytoplasmic vacuolization can be observed in the hepatocytes of mice treated with ritonavir. In conclusion, the mechanism of ritonavir’s hepatotoxicity is that it induces apoptosis of hepatocytes via the caspase-cascade system.

Effect of the disruption of three cytoskeleton components on chondrocyte metabolism in rabbit knee cartilage.

Abstract: Background Chondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure. Microfilaments, microtubules, and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes. The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro. Methods Chondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n = 24). The isolated cells were stabilized for three days and then exposed to low, medium, and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest: colchicine for microtubules, acrylamide for intermediate filaments, and cytochalasin D for actin microfilaments. A group of control cells were treated with carrier. Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents. The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3. The mRNA levels of aggrecan (AGG) and type II collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9. Results In the initial drug-dose-response study, there was no significant difference in the vitality of cells treated with 0.1 µmol/L colchicine, 2.5 mmol/L acrylamide, and 10 µg/L cytochalasin D for two days when compared with the control group of cells. The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei. Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three, six, and nine days, and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells. Cellular proliferation, monolayer morphology, and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period. Conclusions The disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis, increased matrix metalloprotease expression, and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures. Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.

Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure

Abstract: Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure. The pathological significance of Rnd3 loss in the transition to heart failure remains unexplored. To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism, we generated Rnd3+/− haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3+/− mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3+/− haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by fasudil treatment partially improved Rnd3+/− mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho-associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double-knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3+/−/ROCK1−/−). Again, genetic deletion of ROCK1 partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype. These data suggest that downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients. Animals with Rnd3 haploinsufficiency are predisposed to hemodynamic stress. Hyperactivation of Rho kinase activity is responsible in part for the apoptotic cardiomyopathy development. Further investigation of ROCK1-independent mechanisms in Rnd3-mediated cardiac remodeling should be the focus for future study.

The Effects of X-Ray Irradiation on the Proliferation and Apoptosis of MCF-7 Breast Cancer Cells

Abstract: To investigate the effects of X-ray irradiation on the proliferation and apoptosis of MCF-7 breast cancer cells; MCF-7 breast cancer cells were irradiated with X-ray. After irradiation, morphological changes and growth inhibition rate of the irradiated cells were observed under an inverted microscope. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the proliferation of the irradiated MCF-7 cells. Transmission electron microscope was used to observe the morphology and ultrastructure of the irradiated MCF-7 cells. Western blotting was used to analyze the expression level of apoptosis-related protein caspase-3. Our results showed, at 48 h after the irradiation (0 Gy and 8 Gy), cells oval in shape, cell shrinkage or swelling and partial formation of debris under inverted microscope; as well as cytoplasmic vacuolization or inspissation, increased electron density of cytoplasm, structural damage of organelles, blurred mitochondrial cristae and chromatin margination ...