Hypertrophic Gene Expression Research Articles

Soluble epoxide hydrolase inhibitor, TUPS, protects against isoprenaline‐induced cardiac hypertrophy

We have previously shown that isoprenaline-induced cardiac hypertrophy causes significant changes in the expression of cytochromes P450 (CYP) and soluble epoxide hydrolase (sEH) genes. Therefore, it is important to examine whether the inhibition of sEH by 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) will protect against isoprenaline-induced cardiac hypertrophy. Male Sprague-Dawley rats were treated with TUPS (0.65 mg kg(-1) day(-1), p.o.), isoprenaline (5 mg kg(-1) day(-1), i.p.) or the combination of both. In vitro H9c2 cells were treated with isoprenaline (100 μM) in the presence and absence of either TUPS (1 μM) or 11,12 EET (1 μM). The expression of hypertrophic, fibrotic markers and different CYP genes were determined by real-time PCR. Isoprenaline significantly induced the hypertrophic, fibrotic markers as well as the heart to body weight ratio, which was significantly reversed by TUPS. Isoprenaline also caused an induction of CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP4A3 and CYP4F4 gene expression and TUPS significantly inhibited this isoprenaline-mediated effect. Moreover, isoprenaline significantly reduced 5,6-, 8,9-, 11,12- and 14,15-EET and increased their corresponding 8,9-, 11,12- and 14,15-dihydroxyeicosatrienoic acid (DHET) and the 20-HETE metabolites. TUPS abolished these isoprenaline-mediated changes in arachidonic acid (AA) metabolites. In H9c2 cells, isoprenaline caused a significant induction of ANP, BNP and EPHX2 mRNA levels. Both TUPS and 11,12-EET significantly decreased this isoprenaline-mediated induction of ANP, BNP and EPHX2. TUPS partially protects against isoprenaline-induced cardiac hypertrophy, which confirms the role of sEH and CYP enzymes in the development of cardiac hypertrophy.

In vitrogeneration of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide-based scaffold

Tissue engineering of a biological osteochondral multilayered construct with a cartilage-interface subchondral bone layer is a key challenge. This study presented a rabbit bone marrow stromal cell (BMSC)/silk fibroin scaffold-based co-culture approach to generate tissue-engineered osteochondral grafts with an interface. BMSC-seeded scaffolds were first cultured separately in osteogenic and chondrogenic stimulation media. The two differentiated pieces were then combined using an RADA self-assembling peptide and subsequently co-cultured. Gene expression, histological and biochemical analyses were used to evaluate the multilayered structure of the osteochondral graft. A complete osteochondral construct with a cartilage-subchondral bone interface was regenerated and BMSCs were used as the only cell source for the osteochondral construct and interface regeneration. Furthermore, in the intermediate region of co-cultured samples, hypertrophic chondrogenic gene markers type X collagen and MMP-13 were found on both chondrogenic and osteogenic section edges after co-culture. However, significant differences gene expression profile were found in distinct zones of the construct during co-culture and the section in the intermediate region had significantly higher hypertrophic chondrocyte gene expression. Biochemical analyses and histology results further supported this observation. This study showed that specific stimulation from osteogenic and chondrogenic BMSCs affected each other in this co-culture system and induced the formation of an osteochondral interface. Moreover, this system provided a possible approach for generating multilayered osteochondral constructs.

The role of aryl hydrocarbon receptor signaling pathway in cardiotoxicity of acute lead intoxication in vivo and in vitro rat model

Lead (Pb2+) is a naturally occurring systemic toxicant heavy metal that affects several organs in the body including the kidneys, liver, and central nervous system. However, Pb2+-induced cardiotoxicity has never been investigated yet and the exact mechanism of Pb2+ associated cardiotoxicity has not been studied. The current study was designed to investigate the potential effect of Pb2+ to induce cardiotoxicity in vivo and in vitro rat model and to explore the molecular mechanisms and the role of aryl hydrocarbon receptor (AhR) and regulated gene, cytochrome P4501A1 (CYP1A1), in Pb2+-mediated cardiotoxicity. For these purposes, Wistar albino rats were treated with Pb2+ (25, 50 and 100mg/kg, i.p.) for three days and the effects on physiological and histopathological parameters of cardiotoxicity were determined. At the in vitro level, rat cardiomyocyte H9c2 cell lines were incubated with increasing concentration of Pb2+ (25, 50, and 100μM) and the expression of hypertrophic genes, α- and β-myosin heavy chain (α-MHC and β-MHC), brain Natriuretic Peptide (BNP), and CYP1A1 were determined at the mRNA and protein levels using real-time PCR and Western blot analysis, respectively. The results showed that Pb2+ significantly induced cardiotoxicity and heart failure as evidenced by increase cardiac enzymes, lactate dehydrogenase and creatine kinase and changes in histopathology in vivo. In addition, Pb2+ treatment induced β-MHC and BNP whereas inhibited α-MHC mRNA and protein levels in vivo in a dose-dependent manner. In contrast, at the in vitro level, Pb2+ treatment induced both β-MHC and α-MHC mRNA levels in time- and dose-dependent manner. Importantly, these changes were accompanied with a proportional increase in the expression of CYP1A1 mRNA and protein expression levels, suggesting a role for the CYP1A1 in cardiotoxicity. The direct evidence for the involvement of CYP1A1 in the induction of cardiotoxicity by Pb2+ was evidenced by the ability of AhR antagonist, resveratrol, to significantly inhibit the Pb2+-modulated effect on β-MHC and α-MHC mRNAs. It was concluded that acute lead exposure induced cardiotoxicity through AhR/CYP1A1-mediated mechanism.

Yes-associated Protein Isoform 1 (Yap1) Promotes Cardiomyocyte Survival and Growth to Protect against Myocardial Ischemic Injury

Yap1 is an important regulator of cardiomyocyte proliferation and embryonic heart development, yet the function of endogenous Yap1 in the adult heart remains unknown. We studied the role of Yap1 in maintaining basal cardiac function and in modulating injury after chronic myocardial infarction (MI). Cardiomyocyte-specific homozygous inactivation of Yap1 in the postnatal heart (Yap(F/F)Cre) elicited increased myocyte apoptosis and fibrosis, dilated cardiomyopathy, and premature death. Heterozygous deletion (Yap(+/F)Cre) did not cause an overt cardiac phenotype compared with Yap(F/F) control mice at base line. In response to stress (MI), nuclear Yap1 was found selectively in the border zone and not in the remote area of the heart. After chronic MI (28 days), Yap(+/F)Cre mice had significantly increased myocyte apoptosis and fibrosis, with attenuated compensatory cardiomyocyte hypertrophy, and further impaired function versus Yap(+/F) control mice. Studies in isolated cardiomyocytes demonstrated that Yap1 expression is sufficient to promote increased cell size and hypertrophic gene expression and protected cardiomyocytes against H(2)O(2)-induced cell death, whereas Yap1 depletion attenuated phenylephrine-induced hypertrophy and augmented apoptosis. Finally, we observed a significant decrease in cardiomyocyte proliferation in Yap(+/F)Cre hearts compared with Yap(+/F) controls after MI and demonstrated that Yap1 is sufficient to promote cardiomyocyte proliferation in isolated cardiomyocytes. Our findings suggest that Yap1 is critical for basal heart homeostasis and that Yap1 deficiency exacerbates injury in response to chronic MI.

Cardiomyocyte-Specific Overexpression of HEXIM1 Prevents Right Ventricular Hypertrophy in Hypoxia-Induced Pulmonary Hypertension in Mice

Right ventricular hypertrophy (RVH) and right ventricular (RV) contractile dysfunction are major determinants of prognosis in pulmonary arterial hypertension (PAH) and PAH remains a severe disease. Recently, direct interruption of left ventricular hypertrophy has been suggested to decrease the risk of left-sided heart failure. Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is a negative regulator of positive transcription elongation factor b (P-TEFb), which activates RNA polymerase II (RNAPII)-dependent transcription and whose activation is strongly associated with left ventricular hypertrophy. We hypothesized that during the progression of PAH, increased P-TEFb activity might also play a role in RVH, and that HEXIM1 might have a preventive role against such process. We revealed that, in the mouse heart, HEXIM1 is highly expressed in the early postnatal period and its expression is gradually decreased, and that prostaglandin I2, a therapeutic drug for PAH, increases HEXIM1 levels in cardiomyocytes. These results suggest that HEXIM1 might possess negative effect on cardiomyocyte growth and take part in cardiomyocyte regulation in RV. Using adenovirus-mediated gene delivery to cultured rat cardiomyocytes, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced phosphorylation of RNAPII, cardiomyocyte hypertrophy, and mRNA expression of hypertrophic genes, whereas a HEXIM1 mutant lacking central basic region, which diminishes P-TEFb-suppressing activity, could not. Moreover, we created cardiomyocyte-specific HEXIM1 transgenic mice and revealed that HEXIM1 ameliorates RVH and prevents RV dilatation in hypoxia-induced PAH model. Taken together, these findings indicate that cardiomyocyte-specific overexpression of HEXIM1 inhibits progression to RVH under chronic hypoxia, most possibly via inhibition of P-TEFb-mediated enlargement of cardiomyocytes. We conclude that P-TEFb/HEXIM1-dependent transcriptional regulation may play a pathophysiological role in RVH and be a novel therapeutic target for mitigating RVH in PAH.

Hypoxia Inhibits Hypertrophic Differentiation and Endochondral Ossification in Explanted Tibiae

PurposeHypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself.Materials and MethodsFetal mouse tibiae (E17.5) explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively). Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion.ResultsThe oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation.ConclusionsCollectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

MAP kinase kinase kinase-2 (MEKK2) regulates hypertrophic remodeling of the right ventricle in hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) results in pressure overload of the right ventricle (RV) of the heart, initiating pathological RV remodeling and ultimately leading to right heart failure. Substantial research indicates that signaling through the MAPK superfamily mediates pathological cardiac remodeling. These considerations led us to test the hypothesis that the regulatory protein MAPKKK-2 (MEKK2) contributes to RV hypertrophy in hypoxia-induced PH. Transgenic mice with global knockout of MEKK2 (MEKK2(-/-) mice) and age-matched wild-type (WT) mice were exposed to chronic hypobaric hypoxia (10% O(2), 6 wk) and compared with animals under normoxia. Exposure to chronic hypoxia induced PH in WT and MEKK2(-/-) mice. In response to PH, WT mice showed RV hypertrophy, demonstrated as increased ratio of RV weight to body weight, increased RV wall thickness at diastole, and increased cardiac myocyte size compared with normoxic control animals. In contrast, each of these measures of RV hypertrophy seen in WT mice after chronic hypoxia was attenuated in MEKK2(-/-) mice. Furthermore, chronic hypoxia elicited altered programs of hypertrophic and inflammatory gene expression consistent with pathological RV remodeling in WT mice; MEKK2 deletion selectively inhibited inflammatory gene expression compared with WT mice. The actions of MEKK2 were mediated in part through regulation of the abundance and phosphorylation of its effector, ERK5. In conclusion, signaling by MEKK2 contributes to RV hypertrophy and altered myocardial inflammatory gene expression in response to hypoxia-induced PH. Therapies targeting MEKK2 may protect the myocardium from hypertrophy and pathological remodeling in human PH.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

THE ST3GAL-IV GENE MEDIATES STRESS-DEPENDENT CARDIAC REMODELLING IN MICE

ObjectivesTo explore the role and mechanisms of ST3Gal-IV gene mediates stress-dependent cardiac remodelling in mice.MethodsBy using transverse aortic constriction (TAC) model, we observed the phenotype of the ST3Gal-IV gene knock-out...

Determining the Absolute Requirement of G Protein–Coupled Receptor Kinase 5 for Pathological Cardiac Hypertrophy

Rationale: Heart failure (HF) is often the end phase of maladaptive cardiac hypertrophy. A contributing factor is activation of a hypertrophic gene expression program controlled by decreased class II histone deacetylase (HDAC) transcriptional repression via HDAC phosphorylation. Cardiac-specific overexpression of G proteinen–coupled receptor kinase-5 (GRK5) has previously been shown to possess nuclear activity as a HDAC5 kinase, promoting an intolerance to in vivo ventricular pressure overload; however, its endogenous requirement in adaptive and maladaptive hypertrophy remains unknown. Objective: We used mouse models with global or cardiomyocyte-specific GRK5 gene deletion to determine the absolute requirement of endogenous GRK5 for cardiac hypertrophy and HF development after chronic hypertrophic stimuli. Methods and Results: Mice with global deletion of GRK5 were subjected to transverse aortic constriction. At 12 weeks, these mice showed attenuated hypertrophy, remodeling, and hypertrophic gene transcription along with preserved cardiac function. Global GRK5 deletion also diminished hypertrophy and related gene expression due to chronic phenylephrine infusion. We then generated mice with conditional, cardiac-specific deletion of GRK5 that also demonstrated similar protection from pathological cardiac hypertrophy and HF after transverse aortic constriction. Conclusions: These results define myocyte GRK5 as a critical regulator of pathological cardiac growth after ventricular pressure overload, supporting its role as an endogenous (patho)-physiological HDAC kinase. Further, these results define GRK5 as a potential therapeutic target to limit HF development after hypertrophic stress.

A high leucine diet mitigates cardiac injury and improves survival after acute myocardial infarction

ObjectiveEvidence suggests that branched chain amino acids (BCAAs) are beneficial in treating human disease. It is unknown, however, what impact BCAAs have on outcomes in acute myocardial infarction (MI). This study was performed to test the hypothesis that the specific BCAA leucine mitigates cardiac injury and improves survival post-MI. Materials/Methods11–12week old male C57BL/6 mice were subjected to experimental MI or sham procedure, and provided regular chow (RC; 1.5% leucine) or a high leucine diet (HLD; 5% leucine), and followed for 3½ or 28days. All mice were studied by echocardiography and cardiac catheterization, and all hearts were collected for histologic measurements of hypertrophy, fibrosis and apoptosis. Inflammation, hypertrophic gene expression, signal transduction, and glucose, palmitate and leucine metabolism were also measured in 3½day hearts. ResultsExcept for increased leucine and decreased glucose oxidation, an HLD had no effect on measured outcomes in sham mice. With MI, cardiac structure, function, and survival were significantly improved with an HLD. At 3½days post-MI, an HLD increased cardiac hypertrophic signaling and decreased inflammation. Cardiac leucine oxidation was decreased in RC mice post-MI, but significantly increased with an HLD. These changes in metabolism were accompanied by a significant increase in cardiac ATP content in the HLD group. Finally, at both 3½ and 28days post-MI, an HLD increased compensatory hypertrophy, and attenuated cardiac fibrosis and apoptosis. ConclusionsAn HLD increases compensatory hypertrophy, attenuates fibrosis and apoptosis, and positively modulates oxidative metabolism to improve cardiac structure, function, and survival post-MI.

Abstract 111: High Activity Gating of Caveolae-Targeted L-Type Ca 2+ Channels Can Initiate Pathological Hypertrophy

Introduction: The source of Ca 2+ that activates pathological cardiac hypertrophy is not clearly defined. We hypothesize that high activity gating of L-type Ca 2+ channels (LTCCs) localized in caveolae signaling microdomains stabilized by caveolin-3 (Cav-3) provides the Ca 2+ influx that locally activates calcineurin (Cn)-mediated nuclear factor of activated T-cells (NFAT) to induce hypertrophic gene expression. We generated novel reagents that specifically activate or inhibit the gating of LTCCs in caveolae for analysis of the hypertrophic program, as well as any potential effect on ICa and contraction. Methods and Results: We targeted the known LTCC inhibitory protein Rem or an LTCC subunit (β2a) that promotes high activity gating specifically to caveolae by fusing them to a caveolin-binding domain peptide (termed Cav-Rem and Cav-β2a). We infected adult feline left ventricular myocytes (AFLVMs) with adenoviruses containing Cav-Rem or Cav-β2a for membrane localization and functional studies. NFAT nuclear translocation was determined by co-infecting AFLVMs with ad-NFAT-GFP and either ad-Cav-Rem or ad-Cav-β2a and pacing cells to induce Ca2+ influx mediated nuclear NFAT-GFP translocation. Membrane fractionation experiments showed that Cav-3 membrane domains contain 26.2 +/- 12.7% of membrane targeted LTCCs and blocking these with Cav-Rem eliminated a small fraction of the LTCC current (&lt;15%) and almost all Ca2+ influx induced NFAT nuclear translocation (&gt;90%), but did not reduce myocyte contractility. Conversely, selective enhancement of LTCC activity within caveolae with Cav-β2a caused a significant increase in NFAT nuclear translocation (&gt;70%) but had no significant effect on contractility. Conclusions: We provide proof of concept that specific Cav-targeted reagents can be used to enhance or inhibit LTCC activity within caveolae microdomains to amplify or block the hypertrophic response. Our results suggest that high activity gating of LTCCs, which is known to be present in the hypertrophied failing human heart, can activate signaling pathways linked to cardiac hypertrophy. Selectively inhibiting these caveolae localized LTCCs could be a novel mechanism to block pathological hypertrophy without reducing cardiac contractility.

Highlights from the Literature

Highlights from the Literature

Na+/H+ Exchanger 1 Directly Binds to Calcineurin A and Activates Downstream NFAT Signaling, Leading to Cardiomyocyte Hypertrophy

The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.

Calmodulin-Dependent Protein Kinase II: Linking Heart Failure and Arrhythmias

Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled over the past decade supports a view that activation of the multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca(2+) or oxidation, upstream signals with the capacity to transition CaMKII into a Ca(2+) and calmodulin-independent constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation-contraction coupling and cell survival, including ion channels and Ca(2+) homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof-of-concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and for reducing arrhythmias. We review the molecular physiology of CaMKII and discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction, and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.

The α1A‐adrenergic receptor mediates cardiac hypertrophy through the Gαq‐PI3K‐Rac1 signaling pathway

Using a mouse model of cardiac‐specific over‐expression of the α1A‐adrenergic receptor (α1A‐AR; α1A‐M 66‐fold; α1A‐H 166‐fold) we have recently shown that acute activation of the α1A‐AR results in enhanced contraction through activation of α1A‐AR‐operated Ca2+ entry via Snapin and TRPC6.We have now defined a hypertrophic signalling pathway involving α1A‐AR‐mediated recruitment of Gαq‐PI3K‐Rac1 that is distinct from the contractile pathway. Cardiomyocytes from adult α1A‐M, α1A‐H and WT mice stimulated with the α1A‐AR‐specific agonist A61603 exhibited dose‐dependent increases in hypertrophic gene expression, protein synthesis, and cell surface area that were proportional to the level of receptor expression and were Gαq‐PI3K‐Rac1‐dependent. Inhibition of the α1A‐AR contractile pathway by blockade of TRPC6 did not inhibit α1A‐AR‐mediated induction of cardiac hypertrophy.The elucidation of two distinct α1A‐AR‐mediated signalling pathways, one increasing cardiac contraction upon acute activation and the other inducing cardiac hypertrophy upon chronic activation, identifies the α1A‐AR as a potential target for the development of pathway‐specific α1A‐AR‐agonist drugs to treat heart failure.Sponsor: NHMRC, NHF

Reversible cardiac remodeling after renin‐angiotensin system stimulation in CYP1A1‐Ren2 transgenic rats

In transgenic inducible Cyp1a1‐Ren2 hypertensive rats (IHR) (pro)renin levels and blood pressure (BP) can be dose‐dependently titrated by administration of indole‐3‐carbinol (I3C). We examined if activation of (pro)renin to levels that cause severe hypertension (HT) would provoke hypertrophic cardiomyopathy or heart failure (HF). Echocardiographic studies in young and adult IHR revealed that stimulation of (pro)renin for 4 weeks (SBP&gt;200mmHg) did not result in HF but incited a cardiac remodeling process characterized by increased left ventricular (LV) wall thickness and decreased LV volume. Hypertrophic gene expression was upregulated, whereas activation of matricellular, inflammatory and fibrotic responses were absent. We tested also if this cardiac remodeling process could be reversed by de‐activation of the (pro)renin system. Four weeks after withdrawal of I3C, (pro)renin levels were normalized young and adult IHR. While in adult IHR BP returned to normal, HT was sustained in young IHR. Despite the latter, myocardial hypertrophy was fully regressed in both young and adult IHR. We conclude that (pro)renin induced severe HT in IHR causes an age‐independent fully reversible myocardial concentric hypertrophic remodeling. In addition, de‐activation of the renin‐angiotensin system was sufficient for the reversion of cardiac hypertrophy, despite the presence of elevated BP.

Investigation of chondrocyte hypertrophy and cartilage calcification in a full-depth articular cartilage explants model

Articular cartilage deterioration, which includes cartilage degradation and chondrocyte hypertrophy, is a hallmark of degenerative joint diseases (DJD). Chondrocyte hypertrophy is initiated in the deep layer of the cartilage; thus, a robust explants model for investigation of hypertrophy should include this zone. The aim of this study was to characterize and investigate the hypertrophy-promoting potential of different endogenous factors on an ex vivo articular cartilage model. The full-depth cartilage explants were harvested from bovine femoral condyle and cultured for 13 days in different conditions: 10 ng/ml oncostatin M + 20 ng/ml TNF-α; 100 ng/ml IGF1; 10-100 ng/ml bFGF; 10-100 ng/ml BMP2; 50 μg/ml ascorbic acid in combination with 10 mM β-glycerophosphate; and 20-100 ng/ml triiodothyronine. The cellular activity and morphology, degradation, formation and calcification, and expression level of hypertrophic markers were investigated. The hypertrophic factors tested all induced cellular activity and marked morphological changes starting at day 4, however, not in a synchronized manner. Both cartilage degradation and formation were induced by T3 (P < 0.05). Only T3 had a full hypertrophic gene expression profile (P < 0.05). We developed and characterized a novel model for investigation of chondrocyte hypertrophy. We speculated that this can become an important investigatory tool for investigation of matrix turnover, chondrocyte hypertrophy and cartilage calcification that are associated with DJD pathogenesis.

Structured three‐dimensional co‐culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy

Menisci play a crucial role in weight distribution, load bearing, shock absorption, lubrication, and nutrition of articular cartilage within the knee joint. Damage to the meniscus typically does not heal spontaneously due to its partial avascular nature. Partial or complete meniscectomy is a common clinical treatment of the defective meniscus. However, this procedure ultimately leads to osteoarthritis due to increased mechanical stress to the articular cartilage. Meniscus tissue engineering offers a promising solution for partial or complete meniscus deficiency. Mesenchymal stem cells (MSC) have the potential to differentiate into meniscal fibrochondrocyte as well as deliver trophic effects to the differentiated cells. This study tested the feasibility of using MSC co-cultured with mature meniscal cells (MC) for meniscus tissue engineering. Structured cell pellets were created using MC and MSC at varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) and cultured with or without transforming growth factor-beta 3 supplemented chondrogenic media for 21 days. The meniscal and hypertrophic gene expression, gross appearance and structure of the pellets, meniscus extracellular matrix (ECM), histology and immunohistochemistry of proteoglycan and collagen were evaluated. Co-culture of MC with MSC at 75:25 demonstrated highest levels of collagen type I and glycosaminoglycans (GAG) production, as well as the lowest levels of hypertrophic genes, such as COL10A1 and MMP13. All co-culture conditions showed better meniscus ECM production and hypertrophic inhibition as compared to MSC culture alone. The collagen fiber bundles observed in the co-cultures are important to produce heterogenic ECM structure of meniscus. In conclusion, co-culturing MC and MSC is a feasible and efficient approach to engineer meniscus tissue with enhanced ECM production without hypertrophy.

Cardiac remodeling is not modulated by overexpression of muscle LIM protein (MLP)

Muscle LIM protein (MLP) has been proposed to be a central player in the pathogenesis of heart muscle disease. In line with this notion, the homozygous loss of MLP results in cardiac hypertrophy and dilated cardiomyopathy. Moreover, MLP is induced in several models of cardiac hypertrophy such as aortic banding and myocardial infarction. We thus hypothesized that overexpression of MLP might change the hypertrophic response to cardiac stress. In order to answer the question whether MLP modulates cardiac hypertrophy in vivo, we generated a novel transgenic mouse model with cardiac-specific overexpression of MLP. Three independent transgenic lines did not show a pathological phenotype under baseline conditions. Specifically, contractile function and heart weight to body weight ratios at different ages were normal. Next, the transgenic animals were challenged with pressure overload due to aortic constriction. Surprisingly, transgenic mice developed cardiac hypertrophy to the same extent as their wild-type littermates. Moreover, neither contractile dysfunction nor pathological gene expression in response to pressure overload were differentially affected by MLP overexpression. Finally, in a milder in vivo model of hypertrophy induced by chronic infusion of angiotensin-II, cardiac mass and hypertrophic gene expression were again identical in MLP transgenic mice and controls. Taken together, we provide evidence that cardiac overexpression of MLP does not modulate the heart's response to various forms of pathological stress.