Rick van Leeuwen

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.

Thrombospondin-2 Is Essential for Myocardial Matrix Integrity: Increased Expression Identifies Failure-Prone Cardiac Hypertrophy

Cardiac hypertrophy can lead to heart failure (HF), but it is unpredictable which hypertrophied myocardium will progress to HF. We surmised that apart from hypertrophy-related genes, failure-related genes are expressed before the onset of failure, permitting molecular prediction of HF. Hearts from hypertensive homozygous renin-overexpressing (Ren-2) rats that had progressed to early HF were compared by microarray analysis to Ren-2 rats that had remained compensated. To identify which HF-related genes preceded failure, cardiac biopsy specimens were taken during compensated hypertrophy and we then monitored whether the rat progressed to HF or remained compensated. Among 48 genes overexpressed in failing hearts, we focused on thrombospondin-2 (TSP2). TSP2 was selectively overexpressed only in biopsy specimens from rats that later progressed to HF. Moreover, expression of TSP2 was increased in human hypertrophied hearts with decreased (0.19±0.01) versus normal ejection fraction (0.11±0.03 [arbitrary units]; P<0.05). Angiotensin II induced fatal cardiac rupture in 70% of TSP2 knockout mice, with cardiac failure in the surviving mice; this was not seen in wild-type mice. In TSP2 knockout mice, angiotensin II increased matrix metalloproteinase (MMP)-2 and MMP-9 activity by 120% and 390% compared with wild-type mice (P<0.05). In conclusion, we identify TSP2 as a crucial regulator of the integrity of the cardiac matrix that is necessary for the myocardium to cope with increased loading and that may function by its regulation of MMP activity. This suggests that expression of TSP2 marks an early-stage molecular program that is activated uniquely in hypertrophied hearts that are prone to fail.

MicroRNA‐18 and microRNA‐19 regulate CTGF and TSP‐1 expression in age‐related heart failure

To understand the process of cardiac aging, it is of crucial importance to gain insight into the age‐related changes in gene expression in the senescent failing heart. Age‐related cardiac remodeling is known to be accompanied by changes in extracellular matrix (ECM) gene and protein levels. Small noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of ECM proteins. However, their role in age‐related cardiac remodeling and heart failure is unknown. In this study, we investigated the aging‐associated microRNA cluster 17–92, which targets the ECM proteins connective tissue growth factor (CTGF) and thrombospondin‐1 (TSP‐1). We employed aged mice with a failure‐resistant (C57Bl6) and failure‐prone (C57Bl6 × 129Sv) genetic background and extrapolated our findings to human age‐associated heart failure. In aging‐associated heart failure, we linked an aging‐induced increase in the ECM proteins CTGF and TSP‐1 to a decreased expression of their targeting microRNAs 18a, 19a, and 19b, all members of the miR‐17–92 cluster. Failure‐resistant mice showed an opposite expression pattern for both the ECM proteins and the microRNAs. We showed that these expression changes are specific for cardiomyocytes and are absent in cardiac fibroblasts. In cardiomyocytes, modulation of miR‐18/19 changes the levels of ECM proteins CTGF and TSP‐1 and collagens type 1 and 3. Together, our data support a role for cardiomyocyte‐derived miR‐18/19 during cardiac aging, in the fine‐tuning of cardiac ECM protein levels. During aging, decreased miR‐18/19 and increased CTGF and TSP‐1 levels identify the failure‐prone heart.

Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction

The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell–matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC-null infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor β (TGF)–mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-β rescued cardiac rupture in SPARC-null mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

MicroRNA Profiling Identifies MicroRNA-155 as an Adverse Mediator of Cardiac Injury and Dysfunction During Acute Viral Myocarditis

Rationale: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. Objective: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. Methods and Results: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. Conclusions: Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

Imatinib Attenuates End-Organ Damage in Hypertensive Homozygous TGR(mRen2)27 Rats

Imatinib specifically inhibits receptor tyrosine kinase signaling and is clinically used to treat leukemia. Receptor tyrosine kinases not only mediate tumor growth but also initiate adverse signaling in heart failure. We investigated whether imatinib, by inhibiting the platelet-derived growth factor receptor-β (PDGFRβ), prevents cardiac and renal damage in TGR(mRen2)27 (Ren2) rats. Eight-week-old male homozygous Ren2 and Sprague Dawley rats were treated either with imatinib (30 mg/kg; STI-571) or placebo for 8 weeks (Ren2 n=12 for each group; Sprague Dawley n=6 for each group). Imatinib did not affect blood pressure or left ventricular (LV) hypertrophy in both groups. Imatinib attenuated the decline in fractional shortening (imatinib versus Ren2 placebo 45±4.5% versus 32±3%; n=7–11; P<0.05) and in diastolic function in Ren2 rats (baseline diastolic dP/dt corrected for systolic blood pressure Ren2 imatinib versus Ren2 placebo 38.6±0.67 versus 35.3±0.41 [1 · s−1]; n=7–11; P<0.05). This was associated with decreased cardiac fibrosis and decreased activation of PDGFRβ and extracellular signal-regulated kinase 1/2. Renal microvascular hypertrophy and perivascular fibrosis in Ren2 rats were significantly decreased by imatinib. In vitro, imatinib blocked angiotensin II-induced activation of the PDGFRβ and significantly decreased fibroblast proliferation and collagen production. In conclusion, imatinib did not affect LV hypertrophy but attenuated the decline in cardiac function and reduced renal microvascular damage associated with reduced activation of the PDGFRβ. The simultaneous improvement in both heart and kidneys suggests that inhibition of the PDGFRβ has broad protective effects that may provide novel avenues for a blood pressure-independent protection against end-organ damage.

N -Acetyl-Ser-Asp-Lys-Pro Inhibits Phosphorylation of Smad2 in Cardiac Fibroblasts

N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a specific substrate for the N-terminal site of ACE and increases 5-fold during ACE inhibitor therapy. It is known to inhibit the proliferation of hematopoietic stem cells and has also recently been reported to inhibit the growth of cardiac fibroblasts. We investigated its mode of action in cardiac fibroblasts by assessing its influence on transforming growth factor &bgr;1 (TGF&bgr;1)–mediated Smad signaling. AcSDKP inhibited the proliferation of isolated cardiac fibroblasts (P <0.05) but significantly stimulated the proliferation of vascular smooth muscle cells. Flow cytometry of rat cardiac fibroblasts treated with AcSDKP showed significant inhibition of the progression of cells from G0/G1 phase to S phase of the cell cycle. In cardiac fibroblasts transfected with a Smad-sensitive luciferase reporter construct, AcSDKP decreased luciferase activity by 55±9.7% (P =0.01). Moreover, phosphorylation and nuclear translocation of Smad2 was decreased in cardiac fibroblasts treated with AcSDKP. To conclude, AcSDKP inhibits the growth of cardiac fibroblasts and also inhibits TGF&bgr;1-stimulated phosphorylation of Smad2. Because AcSDKP increases substantially during ACE inhibitor therapy, this suggests a novel pathway independent of angiotensin II, by which ACE inhibitors can inhibit cardiac fibrosis.

Syndecan-1 Amplifies Angiotensin II–Induced Cardiac Fibrosis

Syndecan-1 (Synd1) is a transmembrane heparan sulfate proteoglycan that functions as a coreceptor for various growth factors and modulates signal transduction. The present study investigated whether Synd1, by affecting growth factor signaling, may play a role in hypertension-induced cardiac fibrosis and dysfunction. Expression of Synd1 was increased significantly in mouse hearts with angiotensin II–induced hypertension, which was spatially related to cardiac fibrosis. Angiotensin II significantly impaired fractional shortening and induced cardiac fibrosis in wild-type mice, whereas these effects were blunted in Synd1-null mice. Angiotensin II significantly increased cardiac expression of connective tissue growth factor and collagen type I and III in wild-type mice, which was blunted in Synd1-null mice. These findings were confirmed in vitro, where angiotensin II induced the expression of both connective tissue growth factor and collagen I in fibroblasts. The absence of Synd1 in either Synd1-null fibroblasts, after knockdown of Synd1 by short hairpin RNA, or after inhibition of heparan sulfates by protamine attenuated this increase, which was associated with reduced phosphorylation of Smad2. In conclusion, loss of Synd1 reduces cardiac fibrosis and dysfunction during angiotensin II–induced hypertension.

Osteoglycin Prevents Cardiac Dilatation and Dysfunction After Myocardial Infarction Through Infarct Collagen Strengthening

Rationale: To maintain cardiac mechanical and structural integrity after an ischemic insult, profound alterations occur within the extracellular matrix. Osteoglycin is a small leucine-rich proteoglycan previously described as a marker of cardiac hypertrophy. Objective: To establish whether osteoglycin may play a role in cardiac integrity and function after myocardial infarction (MI). Methods and Results: Osteoglycin expression is associated with collagen deposition and scar formation in mouse and human MI. Absence of osteoglycin in mice resulted in significantly increased rupture-related mortality with tissue disruption, intramyocardial bleeding, and increased cardiac dysfunction, despite equal infarct sizes. Surviving osteoglycin null mice had greater infarct expansion in comparison with wild-type mice because of impaired collagen fibrillogenesis and maturation in the infarcts as revealed by electron microscopy and collagen polarization. Absence of osteoglycin did not affect cardiomyocyte hypertrophy in the remodeling remote myocardium. In cultured fibroblasts, osteoglycin knockdown or supplementation did not alter transforming growth factor-&bgr; signaling. Adenoviral overexpression of osteoglycin in wild-type mice significantly improved collagen quality, thereby blunting cardiac dilatation and dysfunction after MI. In osteoglycin null mice, adenoviral overexpression of osteoglycin was unable to prevent rupture-related mortality because of insufficiently restoring osteoglycin protein levels in the heart. Finally, circulating osteoglycin levels in patients with heart failure were significantly increased in the patients with a previous history of MI compared with those with nonischemic heart failure and correlated with survival, left ventricular volumes, and other markers of fibrosis. Conclusions: Increased osteoglycin expression in the infarct scar promotes proper collagen maturation and protects against cardiac disruption and adverse remodeling after MI. In human heart failure, osteoglycin is a promising biomarker for ischemic heart failure.