Cardiac Progenitor Cell–Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation

Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI

A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study

Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal antibody infliximab (IFX). The applied chromatographic method utilizes sequential flows of diluted serum, wash buffer, and an immunoglobulin generic label on LF strips with a Test line comprised of TNF-α. The successive flows permitted enrichment of IFX at the Test line before the label was applied. The label, luminescent upconverting phosphor (UCP) particles coated with protein-A, emits a 550-nm visible light upon excitation with 980-nm infrared light. IFX concentrations were determined through measurement of UCP fluorescence at the Test line. The assay was optimized to detect IFX levels as low as 0.17 μg/mL in serum. For patients with IBD, this limit is appropriate to detect levels associated with loss of response (0.5 μg IFX/mL). The assay was evaluated with clinical samples from patients with Crohn's disease and correlated well within the physiologically relevant range from 0.17 to 10 μg/mL with an IFX-specific ELISA. Performance of the assay was further successfully validated with samples from blood donors, IFX negative IBD patients, and rheumatoid arthritis patients that had developed anti-IFX antibodies. Because of its generic nature, the assay is suited for detecting most therapeutic anti-TNF-α monoclonal antibodie

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction

<div><p>Aims</p><p>Hereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFβ co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery.</p><p>Methods and results</p><p>After inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (<i>Eng</i>^+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4⁺ MNCs residing in the infarcted <i>Eng</i>^+/- hearts (<i>Eng</i>^+/- 73.17±12.67 vs. <i>Eng</i>^+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (<i>Eng</i>^+/- 46.60±9.33% vs. <i>Eng</i>^+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in <i>Eng</i>^+/- mice. An increased number of capillaries (<i>Eng</i>^+/- 61.63±1.43 vs. <i>Eng</i>^+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (<i>Eng</i>^+/- 11.88±0.63 vs. <i>Eng</i>^+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in <i>Eng</i>^+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. <i>Eng</i>^+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels.</p><p>Conclusions</p><p>In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in <i>Eng</i>^+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.</p></div

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFβ/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/− mice, a model for HHT1. Eng+/− mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206−) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFβ-induced differentiation of Eng+/− monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/− mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/− mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/− mice

DipA treatment does not maintain improved cardiac function of <i>Eng</i>^<i>+/-</i> mice after MI.

<p><b>(A)</b> Experimental overview and percentage EF at 7 and 14 days post-MI of control treated mice, measured by ultrasound via left ventricle tracing (n = 5–9). Data shown are mean ± SEM, *P<0.05. <b>(B)</b> Percentage EF 7 and 14 days post-MI of DipA treated mice, measured by ultrasound via left ventricle tracing. Note that the control WT and <i>Eng</i>^<i>+/-</i> groups are the repeat of measurements used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189805#pone.0189805.g001" target="_blank">Fig 1D</a> (n = 9–11). Data shown are mean ± SEM, *P<0.05. <b>(C)</b> Long term treatment overview and Δï. DipA treatment (shown in green) up to 14 days post-MI and cardiac function with extended follow-up of 6 months (n = 5–11). Data depicted as ΔEF are the EF at the time point indicated on the x-axis compared to EF measured at day 7 post-MI. Cardiac function was measured by ultrasound via left ventricle tracing. DipA = DPP4 inhibitor Diprotin A, US = Ultrasound measurement. Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p

The BMP Receptor 2 in Pulmonary Arterial Hypertension: When and Where the Animal Model Matches the Patient

Background: Mutations in bone morphogenetic protein receptor type II (BMPR2) are leading to the development of hereditary pulmonary arterial hypertension (PAH). In non-hereditary forms of PAH, perturbations in the transforming growth factor-β (TGF-β)/BMP-axis are believed to cause deficient BMPR2 signaling by changes in receptor expression, the activity of the receptor and/or downstream signaling. To date, BMPR2 expression and its activity in the lungs of patients with non-hereditary PAH is poorly characterized. In recent decades, different animal models have been used to understand the role of BMPR2 signaling in PAH pathophysiology. Specifically, the monocrotaline (MCT) and Sugen-Hypoxia (SuHx) models are extensively used in interventional studies to examine if restoring BMPR2 signaling results in PAH disease reversal. While PAH is assumed to develop in patients over months or years, pulmonary hypertension in experimental animal models develops in days or weeks. It is therefore likely that modifications in BMP and TGF-β signaling in these models do not fully recapitulate those in patients. In order to determine the translational potential of the MCT and SuHx models, we analyzed the BMPR2 expression and activity in the lungs of rats with experimentally induced PAH and compared this to the BMPR2 expression and activity in the lungs of PAH patients. Methods: the BMPR2 expression was analyzed by Western blot analysis and immunofluorescence (IF) microscopy to determine the quantity and localization of the receptor in the lung tissue from normal control subjects and patients with hereditary or idiopathic PAH, as well as in the lungs of control rats and rats with MCT or SuHx-induced PAH. The activation of the BMP pathway was analyzed by determining the level and localization of phosphorylated Smad1/5/8 (pSmad 1/5/8), a downstream mediator of canonical BMPR2 signaling. Results: While BMPR2 and pSmad 1/5/8 expression levels were unaltered in whole lung lysates/homogenates from patients with hereditary and idiopathic PAH, IF analysis showed that BMPR2 and pSmad 1/5/8 levels were markedly decreased in the pulmonary vessels of both PAH patient groups. Whole lung BMPR2 expression was variable in the two PAH rat models, while in both experimental models the expression of BMPR2 in the lung vasculature was increased. However, in the human PAH lungs, the expression of pSmad 1/5/8 was downregulated in the lung vasculature of both experimental models. Conclusion: BMPR2 receptor expression and downstream signaling is reduced in the lung vasculature of patients with idiopathic and hereditary PAH, which cannot be appreciated when using human whole lung lysates. Despite increased BMPR2 expression in the lung vasculature, the MCT and SuHx rat models did develop PAH and impaired downstream BMPR2-Smad signaling similar to our findings in the human lung

Monocyte specific knock-out of endoglin does not recapitulate the <i>Eng</i>^<i>+/-</i> phenotype.

<p><b>(A)</b> Western blot analysis of endoglin protein expression in LysM-Cre-<i>Eng</i>^+/+, LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl cultured macrophages. A representative experiment is shown. <b>(B)</b> Quantification of the Western blots for endoglin protein in LysM-Cre-<i>Eng</i>^+/+, LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl cultured macrophages in two independent experiments(macrophage cultures from 3 individual mice of each genotype were pooled per western blot). <b>(C)</b> Kaplan-Meier survival curve of wild type (WT), LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl mice 28 days post-MI (n = 6–10). <b>(D)</b> Cardiac function in percentage ejection fraction (ï) 14 days post-MI. Cardiac function was measured by ultrasound in long axis view (n = 5–9). Data are shown as mean ± SEM, *P<0.05.</p

DPP4 inhibitor treatment reduces infarct size.

<p><b>(A)</b> Experimental protocol and treatment overview. At day 0, MI is induced and DPP4 inhibition is started (shown in green) till day 5 post-MI by intraperitoneal (i.p.) injection of DipA (treatment group) or distilled water (control group). Cardiac echography was performed at day 7 and 14 post-MI. <b>(B)</b> Histological analysis of the infarct size by Picrosirius red staining for collagen (n = 5–9). Transverse sections of left ventricle, photos taken at 1.0x magnification. Infarct area = dark pink, healthy myocardium = light pink, blood cells = yellow. <b>(C)</b> Quantification of Picrosirius red staining in left ventricle (LV). Mice were subjected to MI and treated with either distilled water or DipA from day 0 till day 5 by daily i.p. injection (n = 5–9). Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p