Mesenchymal stem cells (MSCs) are known for their immunomodulatory functions. We previously demonstrated that bone marrow–derived MSCs effectively control transplant arteriosclerosis (TA) by enhancing IL-10+ and IFN-γ+ cells. The objective of this study is to elucidate the mechanism by which MSCs induce IL-10+IFN-γ+CD4+ regulatory T type 1 (TR1)–like cells. In an MLR system using porcine PBMCs, MSC-induced IL-10+IFN-γ+CD4+ cells, which confer resistance to allogeneic proliferation in an IL-10–dependent manner, resemble TR1-like cells. Both cyclooxygenase-derived PGE2 and IDO help to induce TR1-like cells by MSCs. MSCs constitutively secrete PGE2, which is augmented in allogeneic reactions. However, TR1-like cells were deficient in PGE2 and 4-fold less potent than were MSCs in suppressing MLR. PGE2 mimetic supplements can enhance the immunosuppressive potency of TR1-like cells. In a porcine model of allogeneic femoral arterial transplantation, MSC-induced TR1-like cells combined with PGE2, but not either alone, significantly reduced TA at the end of 6 wk (percentage of luminal area stenosis: TR1-like cells + PGE2: 11 ± 10%; PGE2 alone: 93 ± 8.7%; TR1-like cells alone: 88 ± 2.4% versus untreated 94 ± 0.9%, p < 0.001). These findings indicate that PGE2 helps MSC-induced IL-10+IFN-γ+CD4+ TR1-like cells inhibit TA. PGE2 combined with MSC-induced TR1-like cells represents a new approach for achieving immune tolerance.

Background—: Endocarditis-inducing streptococci form multilayered biofilms in complex with aggregated platelets on injured heart valves, but the host factors that interconnect and entrap these bacteria-platelet aggregates to promote vegetation formation were unclear. Methods and Results—: In a Streptococcus mutans endocarditis rat model, we identified layers of neutrophil extracellular traps interconnecting and entrapping bacteria-platelet aggregates inside vegetation that could be reduced significantly in size along with diminished colonizing bacteria by prophylaxis with intravascular DNase I alone. The combination of activated platelets and specific immunoglobulin G–adsorbed bacteria are required to induce the formation of neutrophil extracellular traps through multiple activation pathways. Bacteria play key roles in coordinating the signaling through spleen tyrosine kinase, Src family kinases, phosphatidylinositol-3-kinase, and p38 mitogen-activated protein kinase pathways to upregulate the expression of P-selectin in platelets, while inducing reactive oxygen species–dependent citrullination in the arm of neutrophils. Neutrophil extracellular traps in turn serve as the scaffold to further enhance and entrap bacteria-platelet aggregate formation and expansion. Conclusions—: Neutrophil extracellular traps promote and expand vegetation formation through enhancing and entrapping bacteria-platelet aggregates on the injured heart valves. (Circulation. 2015;131:571–581. DOI: 10.1161/CIRCULATIONAHA.114.011432.)

Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P_2Yand P_2Upurinoceptors, rat C₆glioma cells have P₂u purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide‐specific phospholipase C (PI‐PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto‐ATPase inhibitors were studied in these three cell types. In CPAE, suramin, at 3–100 μm, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA₂values of 5.5 ± 0.3 and 4.4 ± 0.4, respectively. Reactive blue, at 1–3 μm, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 μm. PPADS, at 10 μm, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 μm. In contrast, a dose‐dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA₂values of 4.8 ± 0.5 and 5.8 ± 0.7, respectively. In C₆glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. The ecto‐ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto‐ATPase present in CPAE, C₆and RAW 264.7 cells, with IC₅₀values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. This study indicates that PPADS, suramin and reactive blue are ecto‐ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist‐mediated PI responses. Reactive blue is a more potent antagonist than suramin on P_2Y, P_2Uand pyrimidinoceptors, and PPADS is a weak antagonist for P_2Yreceptors.

Homocysteine (Hcy) contributes to atherogenesis and angiostasis by altering the phenotype of arterial endothelial cells (ECs). The present study was aimed at elucidating potential mechanisms by which Hcy can slow EC proliferation and induce EC apoptosis, thereby disrupting endothelial integrity. Given the strong mitogenic and antiapoptotic properties of fibroblast growth factor (FGF)2, we examined whether Hcy can modulate its expression. In cultured human coronary and bovine aortic ECs, Hcy exerted time- and concentration-dependent (0 to 500 μmol/L) reduction of the mRNA and protein levels of FGF2, whereas vascular endothelial growth factor expression was not affected until Hcy reached a proapoptotic 500 μmol/L. By testing a panel of signal transduction inhibitors, we found that the Hcy-induced downregulation of FGF2 was specifically attenuated by pertussis toxin, an inhibitor of Gi protein signaling. Hcy induced cell cycle arrest at the G ₁ /S transition and increased TUNEL-positive apoptotic cells in a graded manner. These effects were effectively counteracted by exogenous FGF2. Reporter gene assays showed that Hcy downregulated FGF2 by transcriptional repression of the gene promoter encompassed in a CpG dinucleotide-rich island. This region was heavily methylated at the cytosine residues by Hcy despite decreased methylation potential ( S -adenosylmethionine to S -adenosylhomocysteine ratio). Normal levels of FGF2 transcription were restored to ECs simultaneously exposed to Hcy and 5-aza-deoxycytidine. We conclude that homocysteine disrupts the growth and survival of ECs through a G protein–mediated pathway associated with altered promoter DNA methylation and the transcriptional repression of FGF2.

Cardiac allograft vasculopathy (CAV) is the major determinant of long-term survival after heart transplantation. We aimed to evaluate the efficacy of PET as a noninvasive way to assess the early stages of CAV. Methods: Twenty-seven consecutive patients (20 men and 7 women; mean age ± SD, 46 ± 12 y) who had normal results on coronary angiography and normal left ventricular systolic function (ejection fraction ≥ 60%) were enrolled at 2.5 ± 2.1 y after transplantation. Myocardial blood flow (MBF) was assessed using dynamic ¹³N-ammonia PET at rest and during adenosine-induced hyperemia, and myocardial perfusion reserve (MPR) was calculated as the ratio of hyperemic MBF to resting MBF. Regional ¹³N-ammonia PET was assessed using a 5-point scoring system. The intravascular ultrasound (IVUS) measurements for the extent of intimal hyperplasia, including plaque volume index (calculated as [total plaque volume/total vessel volume] × 100%) and maximum area of stenosis, were compared with MPR by linear regression analysis. Results: In 27 angiographically normal cardiac transplant recipients, MBF at rest and during adenosine stress and MPR of the left anterior descending artery distribution correlated strongly with the other 2 coronary artery distribution territories (r ≥ 0.97, P < 0.0001). Summed stress score and summed difference score showed a moderate inverse correlation with MPR (r = −0.41 and −0.49, respectively; P < 0.05) but not with IVUS measurements. MPR correlated inversely with plaque volume index (r = −0.40, P < 0.05) but not with maximal luminal stenosis as assessed by IVUS. In addition, MPR and IVUS measurements gradually inversely changed after heart transplantation (all P < 0.05). Conclusion: This study confirms that CAV is a progressive process, diffusely involving the epicardial and microvascular coronary system. Plaque burden as determined by IVUS agrees well with MPR as assessed by PET in recipients with normal coronary angiography results. This finding suggests that dynamic ¹³N-ammonia PET is clinically feasible for the early detection of CAV and can be used as a reliable marker of disease progression.