Jasper A. Koerts

Genetic and Pharmacological Inhibition of Galectin-3 Prevents Cardiac Remodeling by Interfering with Myocardial Fibrogenesis

Background— Galectin-3 has been implicated in the development of organ fibrosis. It is unknown whether it is a relevant therapeutic target in cardiac remodeling and heart failure. Methods and Results— Galectin-3 knock-out and wild-type mice were subjected to angiotensin II infusion (2.5 µg/kg for 14 days) or transverse aortic constriction for 28 days to provoke cardiac remodeling. The efficacy of the galectin-3 inhibitor N-acetyllactosamine was evaluated in TGR(mREN2)27 (REN2) rats and in wild-type mice with the aim of reversing established cardiac remodeling after transverse aortic constriction. In wild-type mice, angiotensin II and transverse aortic constriction perturbations caused left-ventricular (LV) hypertrophy, decreased fractional shortening, and increased LV end-diastolic pressure and fibrosis (P<0.05 versus control wild type). Galectin-3 knock-out mice also developed LV hypertrophy but without LV dysfunction and fibrosis (P=NS). In REN2 rats, pharmacological inhibition of galectin-3 attenuated LV dysfunction and fibrosis. To elucidate the beneficial effects of galectin-3 inhibition on myocardial fibrogenesis, cultured fibroblasts were treated with galectin-3 in the absence or presence of galectin-3 inhibitor. Inhibition of galectin-3 was associated with a downregulation in collagen production (collagen I and III), collagen processing, cleavage, cross-linking, and deposition. Similar results were observed in REN2 rats. Inhibition of galectin-3 also attenuated the progression of cardiac remodeling in a long-term transverse aortic constriction mouse model. Conclusions— Genetic disruption and pharmacological inhibition of galectin-3 attenuates cardiac fibrosis, LV dysfunction, and subsequent heart failure development. Drugs binding to galectin-3 may be potential therapeutic candidates for the prevention or reversal of heart failure with extensive fibrosis.

Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats

Bone marrow-derived cells (BMDC) have been proposed to exert beneficial effects after renal ischemia/reperfusion injury (IRI) by engraftment in the tubular epithelium. However, BMDC can give rise to myofibroblasts and may contribute to fibrosis. BMDC contribution to the renal interstitial myofibroblast population in relation to fibrotic changes after IRI in rats was investigated. A model of unilateral renal IRI (45 min of ischemia) was used in F344 rats that were reconstituted with R26-human placental alkaline phosphatase transgenic BM to quantify BMDC contribution to the renal interstitial myofibroblast population over time. After IRI, transient increases in collagen III transcription and interstitial protein deposition were observed, peaking on days 7 and 28, respectively. Interstitial infiltrates of BMDC and myofibroblasts reached a maximum on day 7 and gradually decreased afterward. Over time, an average of 32% of all interstitial alpha-smooth muscle actin-positive myofibroblasts coexpressed R26-human placental alkaline phosphatase and, therefore, were derived from the BM. BMD myofibroblasts produced procollagen I protein and therefore were functional. The postischemic kidney environment was profibrotic, as demonstrated by increased transcription of TGF-beta and decreased transcription of bone morphogenic protein-7. TGF-beta protein was present predominantly in interstitial myofibroblasts but not in BMD myofibroblasts. In conclusion, functional BMD myofibroblasts infiltrate in the postischemic renal interstitium and are involved in extracellular matrix production.

Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny

AIMS Reciprocal plasticity exists between endothelial and mesenchymal lineages. For instance, mature endothelial cells adopt a smooth muscle-like phenotype through transforming growth factor beta-1 (TGFbeta1)-driven endothelial-to-mesenchymal transdifferentiation (EndMT). Peripheral blood contains circulating endothelial progenitor cells of which the endothelial colony-forming cells (ECFCs) harbour stem cell-like properties. Given the plasticity between endothelial and mesenchymal lineages and the stem cell-like properties of ECFCs, we hypothesized that ECFCs can give rise to smooth muscle-like progeny. METHODS AND RESULTS ECFCs were stimulated with TGFbeta1, after which TGFbeta signalling cascades and their downstream effects were investigated. Indeed, EndMT of ECFCs resulted in smooth muscle-like progeniture. TGFbeta1-driven EndMT is mediated by ALK5 kinase activity, increased downstream Smad2 signalling, and reduced protein levels of inhibitor of DNA-binding protein 3. ECFCs lost expression of endothelial markers and endothelial anti-thrombogenic function. Simultaneously, mesenchymal marker expression was gained, cytoskeletal rearrangements occurred, and cells acquired a contractile phenotype. Transdifferentiated ECFCs were phenotypically stable and self-sustaining and, importantly, showed fibroblast growth factor-2 and angiopoietin-1-mediated pro-angiogenic paracrine properties. CONCLUSION Our study is the first to demonstrate that ECFCs can give rise to smooth muscle-like progeny, with potential therapeutic benefits. These findings further illustrate that ECFCs are highly plastic, which by itself has implications for therapeutical use.

Cell plasticity in wound healing: paracrine factors of M1/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts

BackgroundMacrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs).ResultsHDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production.ConclusionsParacrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair.

Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

AIMS Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress. METHODS AND RESULTS In human atherosclerotic plaques and porcine aortic tissues, myo-endothelial cells were identified, suggestive for EndMT. Flow disturbance by thoracic-aortic constriction in mice similarly showed the presence of myo-endothelial cells specifically in regions exposed to disturbed flow. While uniform laminar shear stress (LSS) was found to inhibit EndMT, endothelial cells exposed to disturbed flow underwent EndMT, in vitro and in vivo, and showed atherogenic differentiation. Gain- and loss-of-function studies using a constitutive active mutant of MEK5 and short hairpins targeting ERK5 established a pivotal role for ERK5 signalling in the inhibition of EndMT. CONCLUSION Together, these data suggest that EndMT contributes to neointimal hyperplasia and induces atherogenic differentiation of endothelial cells. Importantly, we uncovered that EndMT is modulated by shear stress in an ERK5-dependent manner. These findings provide new insights in the role of adverse endothelial plasticity in vascular disease and identify a novel atheroprotective mechanism of uniform LSS, namely inhibition of EndMT.

Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components.

Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue repair. Here, we investigate the combined influence of a pro-angiogenic microenvironment and specific extracellular matrix (ECM) components or tissue culture polystyrene (TCPS) on the dynamics of human macrophage polarization. We established that human angiogenically primed macrophages cultured on different ECM components exhibit an M2-like polarization. These M2-like macrophages polarized to M1 and M2 macrophages with classical (LPS and IFNγ) stimuli and alternative (IL-4 and IL-13) stimuli respectively. Moreover, these M1 and M2 (primary) polarized macrophages rapidly underwent a secondary (re)polarization to M2 and M1 with conditioned media from M2 and M1 primary polarized macrophages respectively. In these initial priming and later (re)polarization processes the soluble factors had a dominant and orchestrating role, while the type of ECM (collagen I, fibronectin, versus tissue culture polystyrene) did not play a crucial role on the polarization of macrophages.

Inhibition of the miR-155 target NIAM phenocopies the growth promoting effect of miR-155 in B-cell lymphoma

Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, NIAM, TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the novel miR-155 target gene NIAM increased proliferation of BL cells. In primary B-cell lymphoma NIAM-positive cases have significant lower levels of miR-155 as compared to NIAM-negative cases, suggesting that NIAM is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate an oncogenic role for miR-155 in B-cell lymphoma which involves targeting the tumor suppressor NIAM.

Tubular engraftment and myofibroblast differentiation of recipient-derived cells after experimental kidney transplantation

Background. In human renal allografts, recipient-derived cells engrafted in various kidney substructures, have been detected in the long term after transplantation. Here we investigated tubular engraftment and myofibroblast differentiation of recipient-derived cells at short term after experimental kidney transplantation, during a previously described window of regeneration and possible onset of renal interstitial fibrosis. Methods. Fisher (F344, syngeneic) and Dark Agouti (DA, allogeneic) kidneys were transplanted into F344-hPAP transgenic recipient rats, which allowed tracing of recipient-derived cells in nontransgenic donor kidneys. We evaluated tubular engraftment and myofibroblast differentiation of recipient-derived cells on day 14 after kidney transplantation. Results. Kidney transplantation resulted in tubular engraftment of recipient-derived cells. After allogeneic kidney transplantation, 9.7% of tubular cross-sections contained at least one recipient-derived cell, which represented a significant increase in comparison to syngeneic transplantation (4.0%, P<0.05). Moreover, recipient-derived myofibroblasts were present in the renal interstitium of the transplanted kidney. These cells contributed 39% of the total interstitial myofibroblast population in allografts, which was comparable to the syngeneic situation (28%, P=0.25). Conclusions. In a defined early window of regeneration and possible onset of renal interstitial fibrosis after kidney transplantation, rejection-associated injury, superimposed on ischemic damage, increases tubular engraftment of recipient-derived cells, although it does not affect their relative contribution to the renal interstitial myofibroblast population.

ZDHHC11 and ZDHHC11B are critical novel components of the oncogenic MYC-miR-150-MYB network in Burkitt lymphoma

ZDHHC11 and ZDHHC11B are critical novel components of the oncogenic MYC-miR-150-MYB network in Burkitt lymphoma

Ciclosporin Does Not Influence Bone Marrow-Derived Cell Differentiation to Myofibroblasts Early after Renal Ischemia/Reperfusion

Background: Ischemia/reperfusion injury (IRI) is a risk factor for the development of interstitial fibrosis. Previously we had shown that after renal IRI, bone marrow-derived cells (BMDC) can differentiate to interstitial myofibroblasts. Here we hypothesized that the immunosuppressant ciclosporin A (CsA), known for its profibrotic side effect, promotes myofibroblast differentiation of BMDC in the postischemic kidney. Methods: Using a model of unilateral renal IRI in rats reconstituted with R26-human placental alkaline phosphatase transgenic bone marrow, CsA was administered in a previously defined critical window for differentiation of BMDC to myofibroblasts. We evaluated fibrotic changes in the kidney and myofibroblast differentiation of BMDC on day 14 after CsA treatment. Results: CsA treatment for 14 days led to increased transforming growth factor-β transcript levels and collagen III deposition in the postischemic kidney. However, neither the total number of α-smooth-muscle-actin-positive interstitial myofibroblasts, nor the bone marrow-derived fraction thereof was affected by CsA administration, irrespective of dosage and duration of treatment. Conclusions: In the critical postischemic window of BMDC differentiation to myofibroblasts, CsA did not promote BMDC differentiation to myofibroblasts, suggesting that, in the clinical setting, CsA is not involved in myofibroblastic differentiation of BMDC.