Ingeborg van der Made

miR-133 and miR-30 Regulate Connective Tissue Growth Factor. Implications for a Role of MicroRNAs in Myocardial Matrix Remodeling

The myocardium of the failing heart undergoes a number of structural alterations, most notably hypertrophy of cardiac myocytes and an increase in extracellular matrix proteins, often seen as primary fibrosis. Connective tissue growth factor (CTGF) is a key molecule in the process of fibrosis and therefore seems an attractive therapeutic target. Regulation of CTGF expression at the promoter level has been studied extensively, but it is unknown how CTGF transcripts are regulated at the posttranscriptional level. Here we provide several lines of evidence to show that CTGF is importantly regulated by 2 major cardiac microRNAs (miRNAs), miR-133 and miR-30. First, the expression of both miRNAs was inversely related to the amount of CTGF in 2 rodent models of heart disease and in human pathological left ventricular hypertrophy. Second, in cultured cardiomyocytes and fibroblasts, knockdown of these miRNAs increased CTGF levels. Third, overexpression of miR-133 or miR-30c decreased CTGF levels, which was accompanied by decreased production of collagens. Fourth, we show that CTGF is a direct target of these miRNAs, because they directly interact with the 3′ untranslated region of CTGF. Taken together, our results indicate that miR-133 and miR-30 importantly limit the production of CTGF. We also provide evidence that the decrease of these 2 miRNAs in pathological left ventricular hypertrophy allows CTGF levels to increase, which contributes to collagen synthesis. In conclusion, our results show that both miR-133 and miR-30 directly downregulate CTGF, a key profibrotic protein, and thereby establish an important role for these miRNAs in the control of structural changes in the extracellular matrix of the myocardium.

Endothelial Cell-Specific NF-κB Inhibition Protects Mice from Atherosclerosis

Atherosclerosis is a progressive disorder of the arterial wall and the underlying cause of cardiovascular diseases such as heart attack and stroke. Today, atherosclerosis is recognized as a complex disease with a strong inflammatory component. The nuclear factor-kappaB (NF-kappaB) signaling pathway regulates inflammatory responses and has been implicated in atherosclerosis. Here, we addressed the function of NF-kappaB signaling in vascular endothelial cells in the pathogenesis of atherosclerosis in vivo. Endothelium-restricted inhibition of NF-kappaB activation, achieved by ablation of NEMO/IKKgamma or expression of dominant-negative IkappaBalpha specifically in endothelial cells, resulted in strongly reduced atherosclerotic plaque formation in ApoE(-/-) mice fed with a cholesterol-rich diet. Inhibition of NF-kappaB abrogated adhesion molecule induction in endothelial cells, impaired macrophage recruitment to atherosclerotic plaques, and reduced expression of cytokines and chemokines in the aorta. Thus, endothelial NF-kappaB signaling orchestrates proinflammatory gene expression at the arterial wall and promotes the pathogenesis of atherosclerosis.

Myeloid Type I Interferon Signaling Promotes Atherosclerosis by Stimulating Macrophage Recruitment to Lesions

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Macrophage secretory phospholipase A2 group X enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology

Secreted phospholipase A2 group X (sPLA2-X) is one of the most potent enzymes of the phospholipase A2 lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA2-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA2-X enzyme activity in vitro and in vivo, by generating sPLA2-X-overexpressing macrophages and transgenic macrophage-specific sPLA2-X mice. Our results show that sPLA2-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2). Moreover, we found that overexpression of active sPLA2-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA2-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA2-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA2-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA2-X in inflammatory lung disease.

The microRNA-15 family inhibits the TGFβ-pathway in the heart

AIMS The overloaded heart remodels by cardiomyocyte hypertrophy and interstitial fibrosis, which contributes to the development of heart failure. Signalling via the TGFβ-pathway is crucial for this remodelling. Here we tested the hypothesis that microRNAs in the overloaded heart regulate this remodelling process via inhibition of the TGFβ-pathway. METHODS AND RESULTS We show that the miRNA-15 family, which we found to be up-regulated in the overloaded heart in multiple species, inhibits the TGFβ-pathway by targeting of TGFBR1 and several other genes within this pathway directly or indirectly, including p38, SMAD3, SMAD7, and endoglin. Inhibition of miR-15b by subcutaneous injections of LNA-based antimiRs in C57BL/6 mice subjected to transverse aorta constriction aggravated fibrosis and to a lesser extent also hypertrophy. CONCLUSION We identified the miR-15 family as a novel regulator of cardiac hypertrophy and fibrosis acting by inhibition of the TGFβ-pathway.

RBM20 Regulates Circular RNA Production From the Titin Gene.

RATIONALE RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules. OBJECTIVE To establish the role of RBM20 in the formation of circRNAs in the heart. METHODS AND RESULTS Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. CONCLUSIONS We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs.

Macrophage-Specific Expression of Mannose-Binding Lectin Controls Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Background— With consideration of the central role of the innate immune system in atherogenesis and mannose-binding lectin (MBL) as an innate regulator of immunity, the role of MBL in experimental and human atherosclerosis was assessed. Methods and Results— With the use of immunohistochemistry and polymerase chain reaction, deposition and gene expression of MBL-A and -C were assessed in murine atherosclerosis from mice deficient for the low-density lipoprotein receptor (LDLR−/−) after 10 or 18 weeks of high-fat feeding. MBL was present and was produced in 10-week-old lesions, whereas deposition and gene expression were minimal after 18 weeks of high-fat feeding and absent in healthy vasculature. Interestingly, deposition of MBL-A and -C differed: MBL-A predominantly localized in upper medial layers, whereas MBL-C was found in and around intimal macrophages. To further study the role of local MBL production by monocytic cells in atherosclerosis, LDLR−/− mice with MBL-A and -C−/− monocytic cells were construed by bone marrow transplantation. Mice carrying MBL-A and -C double deficient macrophages had increased (30%) atherosclerotic lesions compared with wild-type controls (P=0.015) after 10 weeks of high-fat diet. Subsequently, analysis of MBL deposition and gene expression in advanced human atherosclerotic lesions revealed the presence of MBL protein in ruptured but not stable atherosclerotic lesions. Putatively in agreement with murine data, no MBL gene expression could be detected in advanced human atherosclerotic lesions. Conclusions— These results are the first to show that MBL is abundantly present and locally produced during early atherogenesis. Local MBL expression, by myeloid cells, is shown to critically control development of atherosclerotic lesions.

Regulation of Cardiac Gene Expression by KLF15, a Repressor of Myocardin Activity

Pathological forms of left ventricular hypertrophy (LVH) often progress to heart failure. Specific transcription factors have been identified that activate the gene program to induce pathological forms of LVH. It is likely that apart from activating transcriptional inducers of LVH, constitutive transcriptional repressors need to be removed during the development of cardiac hypertrophy. Here, we report that the constitutive presence of Krüppel-like factor 15 (KLF15) is lost in pathological hypertrophy and that this loss precedes progression toward heart failure. We show that transforming growth factor-β-mediated activation of p38 MAPK is necessary and sufficient to decrease KLF15 expression. We further show that KLF15 robustly inhibits myocardin, a potent transcriptional activator. Loss of KLF15 during pathological LVH relieves the inhibitory effects on myocardin and stimulates the expression of serum response factor target genes, such as atrial natriuretic factor. This uncovers a novel mechanism where activated p38 MAPK decreases KLF15, an important constitutive transcriptional repressor whose removal seems a vital step to allow the induction of pathological LVH.

Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development in ApoE-deficient mice

The cellular composition of an atherosclerotic lesion is determined by cell infiltration, proliferation, and apoptosis. The tumor suppressor gene retinoblastoma (Rb) has been shown to regulate both cell proliferation and cell death in many cell types. To study the role of macrophage Rb in the development of atherosclerosis, we used apoE‐deficient mice with a macrophage‐restricted deletion of Rb (Rbdel mice) and control littermates (Rbfl mice). After 12 wk feeding a cholesterol‐rich diet, the Rbdel mice showed a 51% increase in atherosclerotic lesion area with a 39% increase in the relative number of advanced lesions. Atherosclerotic lesions showed a 13% decrease in relative macrophage area and a 46% increase in relative smooth muscle cell area, reflecting the more advanced state of the lesions. The increase in atherosclerosis was independent of in vitro macrophage modified lipoprotein uptake or cytokine production. Whereas macrophage‐restricted Rb deletion did not affect lesional macrophage apoptosis, a clear 2.6‐fold increase in lesional macrophage proliferation was observed. These studies demonstrate that macrophage Rb is a suppressing factor in the progression of atherosclerosis by reducing macrophage proliferation.—Boesten, L. S. M., Zadelaar, A. S. M., van Nieuwkoop, A., Hu, L., Jonkers, J., van de Water, B., Gijbels, M. J. J. van der Made, I., de Winther, M. P. J., Havekes, L. M., van Vlijmen, B. J. M. Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development in ApoE‐deficient mice. FASEB J. 20, E18–E26 (2006)

Cardiomyocyte-specific miRNA-30c over-expression causes dilated cardiomyopathy.

MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.