Expression Of Hypertrophic Markers Research Articles

EGFR Inhibitor Gefitinib Induces Cardiotoxicity through the Modulation of Cardiac PTEN/Akt/FoxO3a Pathway and Reactive Metabolites Formation: In Vivo and in Vitro Rat Studies.

Gefitinib (GEF) is a selective inhibitor of the epidermal growth factor receptor (EGFR) used to treat non-small cell lung cancer. Yet, few cases of cardiotoxicity have been reported. However, the role of the PTEN/Akt/FoxO3a pathway, which mediates GEF anticancer activity, in GEF cardiotoxicity remains unclear. For this purpose, in vitro H9c2 cells and in vivo rat cardiomyocytes were utilized as study models. Treatment of H9c2 cells and Sprague-Dawley rats with GEF significantly induced the expression of hypertrophic and apoptotic markers at mRNA and protein levels with an increased plasma level of troponin. This was accompanied by induction of autophagy and mitochondrial dysfunction in H9c2 cells. Inhibition of cardiac EGFR activity and Akt cellular content of in vitro and in vivo rat cardiomyocytes by GEF increased PTEN and FoxO3a gene expression and cellular content. Importantly, treatment of H9c2 cells with PI3K/Akt inhibitor increased PTEN and FoxO3a mRNA expression associated with potentiation of GEF cardiotoxicity. In addition, by using LC-MS/MS, we showed that GEF is metabolized in the rat heart microsomes into one cyanide- and two methoxylamine-adduct reactive metabolites, where their formation was entirely blocked by CYP1A1 inhibitor, α-naphthoflavone. The current study concludes that GEF induces cardiotoxicity through modulating the expression and function of the cardiac PTEN/AKT/FoxO3a pathway and the formation of CYP1A1-mediated reactive metabolites.

Up‐regulation of SNHG16 induced by CTCF accelerates cardiac hypertrophy by targeting miR‐182‐5p/IGF1 axis

Long non-coding RNA (lncRNA) SNHG16 has been reported to be significant regulators in multiple cancers. However, never has the relationship between it and cardiac hypertrophy been studied until now. In this study, angiotensin II (Ang II)-treated cardiomyocytes isolated from neonatal mice were used as a model of cardiac hypertrophy in vitro. Real-time quantitative polymerase chain reaction was performed to measure the expression of SNHG16, miR-182-5p, and insulin-like growth factor 1 (IGF1). The relationship between SNHG16 and its downstream genes were corroborated by RNA pull-down and luciferase reporter experiments. Western blot was conducted to detect the expression of markers of hypertrophy. The results disclosed that SNHG16 expression was in a high level in the cardiac hypertrophic model. Down-regulation of SNHG16 could decline the expression of hypertrophic markers and reduce cell surface area induced by Ang II. Moreover, SNHG16 was discovered to be activated by transcription factor CCCTC-binding factor. In addition, SNHG16 could enlarge cell surface area and increase the expression of hypertrophic markers by inhibiting miR-182-5p expression in Ang II-treated cardiomyocytes. Finally, overexpression of IGF1 could rescue the effects of silenced SNHG16 on cardiac hypertrophy cells. In brief, our study illustrated that silenced SNHG16 repressed Ang II-imposed cardiac hypertrophy via targeting miR-182-5p/IGF1 axis.

Nasal Chondrocyte-Derived Soluble Factors Affect Chondrogenesis of Cocultured Mesenchymal Stem Cells.

To investigate the effect of soluble factors released from human nasal chondrocytes (NCs) on cocultured human bone marrow mesenchymal stem cells (MSCs) and NC tissue-engineered constructs. Cartilage engineered from pure NCs on a three-dimensional (3D) porous collagen scaffold was cultured indirectly in a Transwell system with cartilage engineered from a direct coculture of human bone marrow-derived MSCs and NCs on a 3D porous collagen scaffold. The soluble factors were measured in the conditioned media from the different chambers of the Transwell system. Engineered cartilage from cocultures exposed to the pure NC construct exhibited reduced chondrogenic potential relative to control constructs, shown by reduced extracellular matrix deposition and increased expression of hypertrophic markers. Analysis of the soluble factors within the conditioned media showed an increase in inflammatory cytokines in the coculture chamber exposed to the pure NC construct. Principal component analysis revealed that the majority of the data variance could be explained by proinflammatory factors and hypertrophic chondrogenesis. In conclusion, our data suggest that inflammatory cytokines derived from NCs reduce the chondrogenic potential of coculture engineered cartilage through the induction of hypertrophic chondrogenesis. Impact statement The use of engineered cartilage from cocultured nasal chondrocytes (NCs) and mesenchymal stem cells for nasal cartilage reconstruction may be problematic. Our data suggest that the soluble factors from surrounding native NCs in the cartilage to be fixed can compromise the quality of the engineered cartilage if used in reconstructive surgery.

Comparison of immunogenic markers of human chondrocytes and chondroprogenitors derived from non-diseased and osteoarthritic articular cartilage

Background: In the field of cartilage repair, cell-based therapy, employing the use of mesenchymal stem cells and chondrocytes maybe a propitious treatment option. Once transferred, success of transplanted cells is determined by their immunogenicity making assessment imperative. Another contender in the field, may be articular chondroprogenitors (CPs) shown to possess chondrogenic potential and reduced expression of hypertrophy markers. Our aim was to assess immunogenic properties of CPs (non-diseased and osteoarthritic (OA)) and compare them with chondrocytes since it may be a good alternative for cell-based therapy and data regarding its immunogenic profile is limited. Methods: Human chondrocytes and CPs from the same cartilage source were isolated. Passage 0 cells characterized by fluorescence-activated cell sorting against human surface antigen were human leucocyte antigen class I (HLA-A2 and HLA-B7), HLA-DR and its costimulatory molecules (CD80 and CD86) and macrophage/monocyte marker (CD14). Results and conclusion: Our observations indicated that CPs isolated from human non-diseased and OA cartilage showed similar immunogenic properties to chondrocytes isolated from the same source with no significant difference in expression. High-to-moderate expression of MHC class I (HLA-A2 and HLA-B7) and moderate-to-low expression of MHC class II (HLA-DR and its co-stimulatory molecules CD80 and CD86) were observed. This is the first report to shed insight on the immunogenic properties of human cartilage-derived progenitors, a potential contender in the field of regenerative therapy for formation of genuine hyaline cartilage.

Valproate Limits Vascular Remodeling and Protects Against Memory Deficits Following Chronic Hypoperfusion

Background and PurposeChronic cerebral hypoperfusion is a risk factor for vascular cognitive impairment. Bilateral carotid artery stenosis (BCAS), a physiologically relevant model of chronic cerebral hypoperfusion, produces damage to the deep white matter of the brain with corresponding deficits in working memory. BCAS increases histone deacetylase (HDAC) activity and global histone acetylation changes in rat brains which may play a role in smooth muscle cell phenotypic changes and vascular remodeling. We tested the hypothesis that treatment of BCAS rats with the HDAC inhibitor, valproate (VPA), limits remodeling and protect rats from memory deficits.MethodsTwenty female Sprague Dawley rats age 16–20 weeks old were subjected to either sham or BCAS surgery and treated with either 300 mg/kg/day VPA or vehicle for eight weeks. Cognitive function was assessed using the novel object recognition test. Rats were subjected to laser speckle imaging to determine cerebral blood flow and then euthanized. Gene expression of HDAC9, ACTA2 and Ki67was measured in the basilar artery using qPCR. All data are reported as the mean ± the standard error (n=5 per group). All experiments were analyzed using one‐way ANOVA with a Bonferoni correction. P‐values of less than 0.05 were considered significant.ResultsAs expected, there was no significant difference in cerebral perfusion between the sham‐vehicle and the BCAS‐vehicle groups at euthanasia. Furthermore, treatment of the rats with VPA had no effect on cerebral perfusion. HDAC expression in the basilar artery of BCAS‐vehicle rats was increased compared to sham‐vehicle rats (1.6 ± 0.2 vs. 1.0 ± 0.1, P=0.0161) and HDAC expression in BCAS‐VPA rats was restored to the sham‐vehicle treated levels (0.9 ± 0.1 vs. 1.0 ± 0.1). Increased ACTA2 expression is a marker for hypertrophy due to hemodynamic stress and was increased in the basilar artery of BCAS‐vehicle rats compared to sham‐vehicle rats (2.3± 0.8 vs. 1.0 ± 0.1, P=0.0494). While VPA had no effect on ACTA2 expression in the sham‐VPA rats, ACTA2 expression in the basilar arteries of BCAS‐VPA rats was restored to the sham‐vehicle levels (0.9 ± 0.3 vs. 1.0 ± 0.1). MKI67 expression was unchanged in BCAS‐vehicle compared to the sham‐vehicle rats while the proliferative marker was increased in the BCAS‐VPA treated rats compared to the sham‐vehicle (3.2 ± 0.7 vs. 1.5 ± 0.4, P=0.0409) and to the BCAS‐vehicle rats (3.2 ± 0.7 vs. 1.3 ± 0.3, P=0.0298). Novel object exploration tests indicate that BCAS‐vehicle rats explored the novel object 29.4 ± 7.1% less than sham‐vehicle rats (P=0.0186) while BCAS‐VPA rats appeared protected from memory impairment and explored the novel object the same amount as the sham‐vehicle rats.Conclusion and future directionsVPA treatment results in decreased expression of hypertrophic markers and is associated with functional restoration of working memory in BCAS rats with white matter injury caused by BCAS. There is a translational potential for clinically used HDAC inhibitors in the treatment of vascular cognitive impairment. Future work will focus on determining sex differences in the response to BCAS and treatment with VPA as well as quantifying white matter injury, and vessel density within the cortex.Support or Funding InformationThe Brain Aneurysm Foundation Research Award

Combination of low intensity electromagnetic field with chondrogenic agent induces chondrogenesis in mesenchymal stem cells with minimal hypertrophic side effects

ABSTRACTBackground: There are different methods to develop in vitro neo-chondral tissues from adipose-derived stem cells (ADSCs). Application of electromagnetic field (EMF) on ADSCs is one of popular approaches, which results in chondrogenesis. If chondrogenic impact of EMF on ADSCs is supposed to be generalized as a protocol in translational medicine field, possible emergence of early or late hypertrophic maturation, mineralization and inflammatory side effects in chondrogenically differentiating ADSCs should be considered.Methods: The advent of chondrogenic and hypertrophic markers by differentiated cells under standard, platelet-rich plasma (PRP)-based or EMF treatments were monitored. Along with monitoring the expressions of chondrogenic markers, inflammatory and hypertrophic markers, VEGF/TNFα secretion, calcium deposition and ALP activity were evaluated.Results: Accordingly, treatment with %5 PRP results in higher GAG production, enhanced SOX9 transcription, lowered TNFα and VEGF secretions compared to other treatments. Although PRP up-regulates miR-146a and miR-199a in early and late stages of chondrogenesis, respectively, application of EMF + PRP down regulates miR-101 and -145 while up-regulates miR-140 and SOX9 expression.Conclusion: Comparing our results with previous reports suggests that presented EMF-ELF in this study with f = 50 Hz, EMF intensity of less than 30 mT, and 5% PRP (v/v), would facilitate chondrogenesis via mesenchymal stem cells with minor inflammation and hypertrophic maturation.Abbreviations: MSCs: mesenchymal stem cells; TGFβ: transforming growth factor-beta; PRP: platelet-rich plasma; ELF-EMF: extremely low-frequency electromagnetic fields; GAGs: glycosaminoglycans; ADSCs: adipose-derived stem cells; VEGF: vascular endothelial growth factor; TNFα: tumor necrosis factor alpha; ALP: alkaline phosphatase

Inhibition of histone demethylase JMJD1C attenuates cardiac hypertrophy and fibrosis induced by angiotensin II

Pathological cardiac hypertrophy is a major risk factor for cardiovascular morbidity and mortality. Histone demethylases (KDMs) are emerging regulators of transcriptional reprograming in cancer, however, their potential role in abnormal heart growth and fibrosis remains largely unknown. The aim of this current study was to examine the role of JMJD1C, an H3K9me2 specific demethylase, in angiotensin II (Ang II) induced cardiac hypertrophy and fibrosis. In this study, we observed that Ang II could increase the expression of JMJD1C detected by Western blot and RT-qPCR in vitro and in vivo. Immunofluorescence staining showed that the treatment of Ang II could increase cardiomyocyte size. RT-qPCR results have shown that Ang II could increase the expression of cell hypertrophic and fibrotic markers in H9c2 cells. Whereas, inhibition of JMJD1C by shRNA and JIB-04, a small molecule histone demethylase inhibitor, significantly reduced Ang II-induced cell hypertrophy, and hypertrophic and fibrotic marker overexpression. Furthermore, cardiomyocyte JMJD1C knockdown decreased Tissue Inhibitor of Metalloproteinases 1 (TIMP1) transcription with pro-fibrotic activity. In conclusion, JMJD1C plays an important role in Ang II-induced cardiac hypertrophy and fibrosis by activating TIMP1 transcription, targeting of JMJD1C may be an effective strategy for the treatment of Ang II-associated cardiac diseases.

Glycyrrhiza glabra (Licorice) root extract attenuates doxorubicin-induced cardiotoxicity via alleviating oxidative stress and stabilising the cardiac health in H9c2 cardiomyocytes

Ethnopharmacological relevanceDoxorubicin (DOX) is an effective anti-neoplastic drug, however; it has downside effects on cardiac health and other vital organs. The herbal remedies used in day to day life may have a beneficial effect without disturbing the health of the vital organs. Glycyrrhiza glabra L. is a ligneous perennial shrub belonging to Leguminosae/Fabaceae/Papilionaceae family growing in Mediterranean region and Asia and widespread in Turkey, Italy, Spain, Russia, Syria, Iran, China, India and Israel. Commonly known as mulaithi in north India, G. glabra has glycyrrhizin, glycyrrhetic acid, isoliquiritin, isoflavones, etc., which have been reported for several pharmacological activities such as anti-demulcent, anti-ulcer, anti-cancer, anti-inflammatory and anti-diabetic. Aim of the studyThe objective of the present study is to investigate the interaction between the molecular factors like PPAR-α/γ and SIRT-1 during cardiac failure arbitrated by DOX under in vitro conditions and role of Glycyrrhiza glabra (Gg) root extract in alleviating these affects. Materials and methodsIn the present study, we have examined the DOX induced responses in H9c2 cardiomyocytes and investigated the role of phytochemical Glycyrrhiza glabra in modulating these affects. MTT assay was done to evaluate the cell viability, Reactive Oxygen Species (ROS)/Reactive Nitrogen Species (RNS) levels, mitochondrial ROS, mitochondrial membrane potential was estimated using fluorescent probes. The oxidative stress in terms of protein carbonylation, lipid peroxidation and DNA damage was detected via spectrophotometric methods and immune-fluorescence imaging. The cardiac markers and interaction between SIRT-1 and PPAR-α/γ was measured using Real-Time PCR, Western blotting and Co-immunoprecipitation based studies. ResultsThe Glycyrrhiza glabra (Gg) extracts maintained the membrane integrity and improved the lipid homeostasis and stabilized cytoskeletal element actin. Gg phytoextracts attenuated aggravated ROS level, repaired the antioxidant status and consequently, assisted in repairing the DNA damage and mitochondrial function. Further, the expression of hypertrophic markers in the DOX treated cardiomyocytes reconciled the expression factors both at the transcriptional and translational levels after Gg treatment. SIRT-1 mediated pathway and its downstream activator PPARs are significant in maintaining the cellular functions. It was observed that the Gg extract allows regaining the nuclear SIRT-1 and PPAR-γ level which was otherwise reduced with DOX treatment in H9c2 cardiomyocytes. The co-immunoprecipitation (Co-IP) documented that SIRT-1 interacts with PPAR-α in the untreated control H9c2 cardiomyocytes whereas DOX treatment interferes and diminishes this interaction however the Gg treatment maintains this interaction. Knocking down SIRT-1 also downregulated expression of PPAR-α and PPAR-γ in DOX treated cells and Gg treatment was able to enhance the expression of PPAR-α and PPAR-γ in SIRT-1 knocked down cardiomyocytes. ConclusionsThe antioxidant property of Gg defend the cardiac cells against the DOX induced toxicity via; 1) reducing the oxidative stress, 2) maintaining the mitochondrial functions, 3) regulating lipid homeostasis and cardiac metabolism through SIRT-1 pathway, and 4) conserving the cardiac hypertrophy and hence preserving the cardiomyocytes health. Therefore, Gg can be recommended as a healthy supplement with DOX towards cancer therapeutics associated cardiotoxicity.

Aliskiren attenuates cardiac dysfunction by modulation of the mTOR and apoptosis pathways.

Aliskiren (ALS) is well known for its antihypertensive properties. However, the potential underlying the molecular mechanism and the anti-hypertrophic effect of ALS have not yet been fully elucidated. The aim of the present study was to investigate the role of ALS in mammalian target of rapamycin (mTOR) and apoptosis signaling using in vivo and in vitro models of cardiac hypertrophy. A rat model of cardiac hypertrophy was induced by isoproterenol treatment (5 mg·kg-1·day-1) for 4 weeks, with or without ALS treatment at 20 mg·kg-1·day-1. The expression of hypertrophic, fibrotic, and apoptotic markers was determined by RT-qPCR. The protein expression of apoptotic markers mTOR and p-mTOR was assessed by western blot analysis. The proliferation of H9C2 cells was monitored using the MTS assay. Cell apoptosis was analyzed using flow cytometry. In vivo, isoproterenol-treated rats exhibited worse cardiac function, whereas ALS treatment reversed these dysfunctions, which were associated with changes in p-mTOR, Bcl-2, Bax, and cleaved caspase-3 expression, as well as the number of apoptotic cells. In vitro, H9C2 cardiomyocyte viability was significantly inhibited and cardiac hypertrophy was induced by Ang II administration, but ALS reversed Ang II-induced H9C2 cardiomyocyte hypertrophy and death. Furthermore, Ang II triggered the activation of the mTOR and apoptosis pathways in hypertrophic cardiomyocytes that were inhibited by ALS treatment. These results indicated that ALS alleviated cardiac hypertrophy through inhibition of the mTOR and apoptosis pathways in cardiomyocytes.

Functional role of hedgehog pathway in osteoarthritis.

The hedgehog signalling pathway is one of the key regulators of metazoan development, and it plays an important role in the regulation of a variety of developmental and physiological processes. But it is aberrantly activated in many human diseases, including osteoarthritis (OA). In this study, we have reviewed the association of hedgehog signalling pathway in the development and progression of OA and evaluated the efforts to target this pathway for the prevention of OA. Usually in OA, activation of hedgehog induces up-regulation of the expression of hypertrophic markers, including type X collagen, increases production of nitric oxide and prostaglandin E2, several matrix-degrading enzymes including matrix metalloproteinase and a disintegrin and metalloproteinase with thrombospondin motifs in human knee joint cartilage leading to cartilage degeneration, and thus contributes in OA. Targeting hedgehog signalling might be a viable strategy to prevent or treat OA. Chemical inhibitors of hedgehog signalling is promising, but they cause severe side effects. Knockdown of HH gene is not an option for OA treatment in humans because it is not possible to delete HH in larger animals. Efficient knockdown of HH achieved by local delivery of small interfering RNA in future studies utilizing large animal OA models might be a more efficient approach for the prevention of OA. However, it remains a major problem to develop one single scaffold due to the different physiological functions of cartilage and subchondral bones possess. More studies are necessary to identify selective inhibitors for efficiently targeting the hedgehog pathway in clinical conditions.

Effect of SKF‑96365 on cardiomyocyte hypertrophy induced by angiotensin II

Angiotensin II (Ang II) is an important bioactive peptide in the renin-angiotensin system, and it can contribute to cell proliferation and cardiac hypertrophy. Dysfunctions in transient receptor potential canonical (TRPC) channels are involved in many types of cardiovascular diseases. The aim of the present study was to investigate the role of the TRPC channel inhibitor SKF-96365 in cardiomyocyte hypertrophy induced by Ang II and the potential mechanisms of SKF-96365. H9c2 cells were treated with different concentrations of Ang II. The expression levels of cardiomyocyte hypertrophy markers and TRPC channel-related proteins were also determined. The morphology and surface area of the H9c2 cells, the expression of hypertrophic markers and TRPC channel-related proteins and the [3H] leucine incorporation rate were detected in the Ang II-treated H9c2 cells following treatment with the TRPC channel inhibitor SKF-96365. The intracellular Ca2+ concentration was tested by flow cytometry. The present results suggested that the surface area of H9c2 cells treated with Ang II was significantly increased compared with untreated H9c2 cells. The fluorescence intensity of α-actinin, the expression of hypertrophic markers and TRPC-related proteins, the [3H] leucine incorporation rate and the intracellular Ca2+ concentration were all markedly increased in the Ang II-treated H9c2 cells but decreased following SKF-96365 treatment. The present results suggested that Ang II induced cardiomyocyte hypertrophy in H9c2 cells and that the TRPC pathway may be involved in this process. Therefore, SKF-96365 can inhibit cardiomyocyte hypertrophy induced by Ang II by suppressing the TRPC pathway. The present results indicated that TRPC may be a therapeutic target for the development of novel drugs to treat cardiac hypertrophy.

Bioactive peptides attenuate cardiac hypertrophy and fibrosis in spontaneously hypertensive rat hearts

Alcalase potato protein hydrolysate (APPH), a nutraceutical food, might an have important role in anti-obesity activity. Recent studies from our lab indicated that APPH treatment had lipolysis stimulating activity and identified was an efficient anti-obesity diet ingredient. In this study we aim to investigate the beneficial effects of pure peptide amino acid sequences (DIKTNKPVIF (DI) and IF) from APPH supplement in the regulation of cardiac hypertrophy and fibrosis on spontaneously hypertensive rats (SHR). We examined hematoxylin and eosin staining, Masson's trichrome staining, echocardiographic parameters, serum parameters, hypertrophy, inflammation and fibrotic marker expression to demonstrate efficacy of bioactive peptides in a SHR model. There was a significant upregulation between SHR and bioactive peptides treated groups in left heart weight (LHW), LHW/WHW, LHW/Tibia, LVIDd, and LVd mass. In addition, the bioactive peptides repress the protein expression of hypertrophy markers (BNP, MYH7), inflammation (TLR-4, p-NFkB, TNF-α, IL-6), and fibrotic markers (uPA, MMP-2, TIMP1, CTGF). In summary, these results indicate that DI and IF bioactive peptides from APPH attenuate cardiac hypertrophy, inflammation and fibrosis in the SHR model.

Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering.

Osteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering.

MicroRNA-200a-3p Is a Positive Regulator in Cardiac Hypertrophy Through Directly Targeting WDR1 as Well as Modulating PTEN/PI3K/AKT/CREB/WDR1 Signaling.

Cardiac hypertrophy is an adaptive expansion of the myocardium due to the overloaded stress of heart. Recently, emerging studies have drawn a conclusion that microRNAs (miRNAs) are involved in myocardial hypertrophy and even heart failure. To figure out the role of microRNA-200a-3p (miR-200a-3p) in cardiac hypertrophy, the in vitro cardiac hypertrophy model was established in H9c2 cells using angiotensin II (Ang-II) as previously described. First of all, we observed a significant increase of miR-200a-3p expression in Ang-II-induced hypertrophic H9c2 cells. Moreover, inhibition of miR-200a-3p dramatically reversed the Ang-II-upregulated expression of hypertrophic markers (atrial natriuretic peptide, brain natriuretic peptide, and β-MHC) and the expanded cell surface area in H9c2 cells. In addition, our results indicated that miR-200a-3p directly targeted both WDR1 and phosphatase and tensin homolog (PTEN). In this regard, miR-200a-3p further activated PI3K/AKT/CREB pathway so as to intensify its negative regulation on WDR1. At length, WDR1 silence, PTEN inhibitor, and PI3K activator recovered the repressive effect of miR-200a-3p suppression on the development of cardiac hypertrophy. Jointly, our study suggested that miR-200a-3p facilitated cardiac hypertrophy by not only directly targeting WDR1 but also through modulating PTEN/PI3K/AKT/CREB/WDR1 signaling, therefore proving novel downstream molecular pathway of miR-200a-3p in cardiac hypertrophy.

Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway.

A major problem with chondrocytes derived in vitro from stem cells is undesired hypertrophic degeneration, to which articular chondrocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. In vitro differentiation of iPSCs into chondrocytes with an AC-phenotype and resistance to hypertrophy has not been demonstrated so far. Here, we present a novel protocol that succeeded in deriving chondrocytes from human iPSCs without using pro-hypertrophic bone-morphogenetic-proteins. IPSC-chondrocytes had a high cartilage formation capacity and deposited two-fold more proteoglycans per cell than adult ACs. Importantly, cartilage engineered from iPSC-chondrocytes had similar marginal expression of hypertrophic markers (COL10A1, PTH1R, IBSP, ALPL mRNAs) like cartilage from ACs. Collagen X was barely detectable in iPSC-cartilage and 30-fold lower than in hypertrophic cartilage derived from mesenchymal stromal cells (MSCs). Moreover, alkaline phosphatase (ALP) activity remained at basal AC-like levels throughout iPSC chondrogenesis, in contrast to a well-known significant upregulation in hypertrophic MSCs. In line, iPSC-cartilage subjected to mineralizing conditions in vitro showed barely any mineralization, while MSC-derived hypertrophic cartilage mineralized strongly. Low expression of Indian hedgehog (IHH) like in ACs but rising BMP7 expression like in MSCs suggested that phenotype stability was linked to the hedgehog rather than the bone morphogenetic protein (BMP) pathway. Taken together, unlimited amounts of AC-like chondrocytes with a high proteoglycan production reminiscent of juvenile chondrocytes and resistance to hypertrophy and mineralization can now be produced from human iPSCs in vitro. This opens new strategies for cartilage regeneration, disease modeling and pharmacological studies.

YQWY decoction reverses cardiac hypertrophy induced by TAC through inhibiting GATA4 phosphorylation and MAPKs.

To investigate the effect of Yiqi Wenyang (YQWY) decoction on reversing cardiac hypertrophy induced by the transverse aortic constriction (TAC). Wistar rats aged 7-8 weeks were subjected to TAC surgery and then randomly divided into 4 groups (n = 5/group): Sham group, TAC group, low-dose group and high dose group. After 16-week intragastric administration of YQWY decoction, the effect of YQWY decoction on alleviating cardiomyocyte hypertrophy was examined by transthoracic echocardiography (TTE), hematoxylin/eosin (HE), wheat germ agglutinin (WGA) staining, enzyme linked immunosorbent assay (ELISA), Western blot (WB), immunohistochemistry (IHC) and immunofluorescence (IF), respectively. The results showed significant differences in left ventricle volume-diastole/systole (LV Vol d/s), N-terminal pro-B-type brain natriuretic peptide (NT-proBNP) (P < 0.01), Ejection Fraction (EF), LV mass and fractional shortening (FS) (P < 0.05) between YQWY-treated group and TAC group. HE and WGA staining showed that treatment with YQWY decoction dramatically prevented TAC-induced cardiomycyte hypertrophy. Moreover, the results of WB, IHC and IF indicated that administration of YQWY could suppress the expressions of cardiac hypertrophic markers, which included the atrial natriuretic peptide (ANP), BNP and myosin heavy chain 7 (MYH7) (P < 0.05) and inhibit phosphorylation of GATA binding protein 4 (P-GATA4) (P < 0.05), phosphorylation of extracellular signal-regulated kinase (P-ERK) (P < 0.05), phosphorylation of P38 mitogen activated protein kinase (P-P38) (P < 0.05) and phosphorylation of c-Jun N-terminal kinase (P-JNK) (P < 0.05). Thus, we concluded that YQWY decoction suppressed cardiomyocyte hypertrophy and reversed the impaired heart function, and the curative effects of YQWY decoction were associated with the decreased phosphorylation of GATA4 and mitogen activated protein kinases (MAPKs), as well as the reduced expression of the downstream targets of GATA4, including ANP, BNP, and MYH7.

Sodium butyrate attenuates angiotensin II-induced cardiac hypertrophy by inhibiting COX2/PGE2 pathway via a HDAC5/HDAC6-dependent mechanism.

Sodium butyrate (NaBu) is reported to play important roles in a number of chronic diseases. The present work is aimed to investigate the effect of NaBu on angiotensin II (Ang II)‐induced cardiac hypertrophy and the underlying mechanism in in vivo and in vitro models. Sprague Dawley rats were infused with vehicle or Ang II (200 ng/kg/min) and orally administrated with or without NaBu (1 g/kg/d) for two weeks. Cardiac hypertrophy parameters and COX2/PGE2 pathway were analysed by real‐time PCR, ELISA, immunostaining and Western blot. The cardiomyocytes H9C2 cells were used as in vitro model to investigate the role of NaBu (2 mmol/L) in inhibition of Ang II‐induced cardiac hypertrophy. NaBu significantly attenuated Ang II‐induced increase in the mean arterial pressure. Ang II treatment remarkably increased cardiac hypertrophy as indicated by increased ratio of heart weight/body weight and enlarged cardiomyocyte size, extensive fibrosis and inflammation, as well as enhanced expression of hypertrophic markers, whereas hearts from NaBu‐treated rats exhibited a significant reduction in these hypertrophic responses. Mechanistically, NaBu inhibited the expression of COX2/PGE2 along with production of ANP and phosphorylated ERK (pERK) stimulated by Ang II in in vivo and in vitro, which was accompanied by the suppression of HDAC5 and HDAC6 activities. Additionally, knocking down the expression of HDAC5 and HDAC6 via gene‐editing strategy dramatically blocked Ang II‐induced hypertrophic responses through COX2/PGE2 pathway. These results provide solid evidence that NaBu attenuates Ang II‐induced cardiac hypertrophy by inhibiting the activation of COX2/PGE2 pathway in a HDAC5/HDAC6‐dependent manner.

Abstract P157: Role of P47 phox in Angiotensin II Induced Cardiac Hypertrophy and Fibrosis in Mice

NADPH oxidase mediated ROS production has been recognized to be an important contributory mechanism for hypertension and left ventricular hypertrophy (LVH). However, the regulatory role of NADPH oxidase mediated superoxide production in Angiotensin II (AngII) induced cardiac fibrosis and macrophage infiltration is still elusive. In the present study, we investigated the role of phagocytic NADPH oxidase enzyme system in cardiac remodelling induced by AngII in young adult (2-3 months old) male mice deficient in p47 phox , a cytosolic subunit of the NADPH oxidase (KO, n=13) and age-matched wild-type littermates (WT, n=9). Animals were infused with AngII pressor dose (1500 ng/kg/min, s.c) or saline via osmotic minipump (Alzet 2002, USA) for 14 consecutive days and then, subjected to echocardiography (VEVO 1100, Canada). Further animals were sacrificed and hearts was harvested for histological studies and gene expression analysis. AngII treatment in WT and KO mice showed prominent alterations in echocardiographic measurements with no changes in E/A, EF and FS. However, KO mice showed aggravated cardiac hypertrophic response (LVAWd: WT, 1.20±0.09 vs. KO, 1.42±0.04, p&lt;0.05; LVIDd: WT, 3.02±0.20 vs. KO, 2.60±0.07, p&lt;0.05; LVPWd: WT, 1.49±0.06 vs. KO, 1.78±0.06, p&lt;0.05). Histologically, AngII infused KO mice also showed increased cardiomyocyte diameter compared to AngII infused WT mice (WT, 388±16.37 vs. KO, 560.3±41.28 μm 2 ; p&lt;0.01 respectively). Further, AngII infused KO mice showed elevated immune cell infiltration, which were positive for Mac-3 staining compared to AngII infused WT mice (WT, 2.61±0.32 vs. KO, 4.09±0.44 %; p&lt;0.05). Besides, AngII infused KO mice showed augmented interstitial fibrosis (WT, 11.11±0 0.67 vs. KO, 20.85± 3.63 %; p&lt;0.05), thick collagen fibres deposition (WT, 28.93± 0.94 vs. KO, 37.60± 2.49 %; p&lt;0.05) compared to AngII infused WT mice. Moreover, AngII infused KO mice also showed upregulated gene expression of hypertrophic and fibrotic markers, including Nppa, Nppb, Acta1, Myh6, TGF-β1, collagen (I and III) and α-sma compared to AngII infused WT mice. Together, these data suggest that NADPH oxidase and its subunit p47 phox play a pivotal role in AngII induced LVH by regulating fibrotic machinery and macrophage infiltration.

Physioxia Has a Beneficial Effect on Cartilage Matrix Production in Interleukin-1 Beta-Inhibited Mesenchymal Stem Cell Chondrogenesis.

Osteoarthritis (OA) is a degenerative condition that involves the production of inflammatory cytokines (e.g., interleukin-1β (IL-1β), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) that stimulate degradative enzymes, matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS) resulting in articular cartilage breakdown. The presence of interleukin-1β (IL-1β) is one reason for poor clinical outcomes in current cell-based tissue engineering strategies for treating focal early osteoarthritic defects. Mesenchymal stem cells (MSCs) are a potential cell source for articular cartilage regeneration, although IL-1β has been shown to inhibit in vitro chondrogenesis. In vivo, articular chondrocytes reside under a low oxygen environment between 2–5% oxygen (physioxia) and have been shown to enhance in vitro MSC chondrogenic matrix content with reduced hypertrophic marker expression under these conditions. The present investigation sought to understand the effect of physioxia on IL-1β inhibited MSC chondrogenesis. MSCs expanded under physioxic (2% oxygen) and hyperoxic (20%) conditions, then chondrogenically differentiated as pellets in the presence of TGF-β1 and either 0.1 or 0.5 ng/mL IL-1β. Results showed that there were donor variations in response to physioxic culture based on intrinsic GAG content under hyperoxia. In physioxia responsive donors, MSC chondrogenesis significantly increased GAG and collagen II content, whilst hypertrophic markers were reduced compared with hyperoxia. In the presence of IL-1β, these donors showed a significant increase in cartilage matrix gene expression and GAG content relative to hyperoxic conditions. In contrast, a set of MSC donors were unresponsive to physioxia and showed no significant increase in matrix production independent of IL-1β presence. Thus, physioxia has a beneficial effect on MSC cartilage matrix production in responsive donors with or without IL-1β application. The mechanisms controlling the MSC chondrogenic response in both physioxia responsive and unresponsive donors are to be elucidated in future investigations.

TAK733 attenuates adrenergic receptor-mediated cardiomyocyte hypertrophy via inhibiting ErkThr188 phosphorylation.

Cardiac hypertrophy is an important risk factor for heart failure. The MEK-ERK axis has been reported as a major regulator in controlling cardiac hypertrophy. TAK733 is a potent and selective MEK inhibitor that suppresses cell growth in a broad range of cell lines. Therefore, we aimed to investigate the anti-hypertrophic effect of TAK733 in cardiomyocytes. Cardiomyocyte hypertrophy was induced with norepinephrine (NE) or phenylepinephrine (PE) using H9c2 cells. To confirm the cardiomyocyte hypertrophy, cell size and protein synthesis were measured and hypertrophy-related gene expression was estimated by reverse transcription polymerase chain reaction. To identify the signaling pathway involved, immunoblot analysis were performed. We observed that NE activated MEK-ERK signaling and increased ANP and BNP expression, resulting in cardiomyocyte hypertrophy. TAK733 significantly reduced cardiomyocyte hypertrophy by regulating NE-induced ERK1/2 and ERKThr188 activation, hypertrophy marker expression, and cardiomyocyte hypertrophy through depression of MEK activity. In addition, we examined that PE-induced cardiomyocyte hypertrophy was also attenuated by TAK733. Here, we report that TAK733 suppressed NE- or PE-induced cardiomyocyte hypertrophy by repressing a crucial component of cardiac hypertrophy-related pathways. These results suggest that TAK733 may be a useful therapeutics for cardiac hypertrophy and warrants further in vivo studies.