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Mesenchymal stem cells (MSCs) possess immunomodulatory properties and may curtail the inflammatory response that characterizes sepsis and other systemic inflammatory states. We aimed to determine whether intravenous infusion of MSCs is associated with reduced inflammation and improved myocardial function in a rat model of endotoxemia. Adult Sprague-Dawley rats were administered saline (vehicle) or LPS (5 mg/kg) via tail vein injection. Treatments, either vehicle or 2 × 10(6) MSCs, were infused 1 h later via tail vein. Animals were randomly assigned to the following groups: (a) vehicle + vehicle (control; n = 6), (b) LPS + vehicle (n = 6), or (c) LPS + MSCs (n = 6). Six hours after induction of endotoxemia, left ventricular ejection fraction (EF) and fractional shortening (FS) was assessed via parasternal short-axis M-mode echocardiography. Hearts and serum were collected for determination of cytokine levels via enzyme-linked immunosorbent assay. Animals injected with LPS + vehicle exhibited depressed cardiac function as indicated by a 26% and 37% reduction in EF and FS from baseline, respectively. Treatment with MSCs was associated with improved cardiac function compared with vehicle treatment as indicated by a reduction in EF and FS of only 10% and 17%, respectively (P < 0.05). Myocardial levels of TNF-α, IL-1β, and IL-6 were elevated in LPS-treated animals versus control. Similarly, serum levels of IL-1β, IL-6, and IL-10 were increased in LPS-treated animals. Treatment with MSCs, however, was associated with significant reductions in serum levels of IL-1β and IL-6 and in myocardial levels of TNF-α, IL-1β, and IL-6. In addition, treatment with MSCs was associated with a further increase in serum IL-10. Infusion of MSCs modulates the systemic inflammatory response and is associated with improved cardiac function during endotoxemia.

Mesenchymal stem cells (MSCs) protect ischemic tissues in part through paracrine growth factor production. IL-6, which is upregulated in the heart during ischemia, has been shown to enhance stem cell proliferation and migration. The effect of IL-6 on MSC paracrine function, however, remains unknown. In addition, TGF-α increases MSC vascular endothelial growth factor (VEGF) production and may share downstream signaling pathways with IL-6 involving ERK, JNK, and PI3K. We hypothesize that cotreatment with IL-6 and TGF-α will result in greater MSC VEGF production than by either treatment alone via these signaling pathways. Murine MSCs were treated with IL-6 (0.05 ng/mL) with or without TGF-α (250 ng/mL) and in combination with inhibitors of ERKI/II, JNK, and PI3K for 24 h. Vascular endothelial growth factor concentrations in the supernatants were measured using enzyme-linked immunosorbent assay. Phosphorylation of ERK, JNK, and PI3K was measured using Western blot analysis. IL-6 increased MSC VEGF production at a dose of 0.05 ng/mL, and the combination of IL-6 and TGF-α (250 ng/mL) increased VEGF production to a greater extent than IL-6 or TGF-α alone. IL-6 induced phosphorylation of ERK, JNK, and PI3K, and inhibition of each suppressed IL-6-induced VEGF production. TGF-α cotreatment overcame VEGF suppression after ERK2 inhibition but not ERK1, JNK, or PI3K. These data suggest that IL-6 stimulates MSC VEGF production alone and additively with TGF-α via ERK-, JNK-, and PI3K-mediated mechanisms. IL-6 and TGF-α cotreatment may be a useful strategy for enhancing MSC VEGF production and cardioprotection during myocardial ischemia.

Toll-like receptors (TLRs) play a key role in innate immune defenses. After activation by foreign pathogens or host-derived molecules, TLRs signal via overlapping or distinct signaling cascades and eventually induce numerous genes involved in a variety of cellular responses. A growing body of evidence suggests that TLR signaling also plays an important role in cardiac ischemia/reperfusion injury. We review our current understanding of the TLR signaling pathways and their roles in the pathophysiology of cardiac ischemia/reperfusion injury, as well as discuss several mechanisms for TLR activation and regulation.

Phosphoinositide-3 kinases (PI3Ks) are critical regulatory proteins in the immunologic defense system against sepsis. The PI3K mechanism helps modulate cellular survival, innate and adaptive immunities, inflammation, nuclear factor-κB transcription, and may, in turn, play a protective role in sepsis. Animal studies confirm its role in the prevention of organ dysfunction and improvement of survival outcomes. Further advances in the understanding of this key immunomodulatory pathway may provide valuable insights into the manipulation of cellular function for therapeutic treatment of sepsis and other inflammatory diseases.

Mesenchymal stem cell (MSC) infusion may reduce myocardial ischemic injury. TNF-alpha is a proinflammatory cytokine produced in large quantities during myocardial ischemia that can exert beneficial or detrimental effects on MSC function by binding to a 55-kd receptor (TNFR1) or a 75-kd receptor (TNFR2) on MSCs. We investigated whether genetic modification with ablation of TNFR1 and/or TNFR2 affects MSC-mediated protection against myocardial ischemic injury. The MSCs were harvested from wild-type mice (WT-MSCs) and knockout mice with ablation of TNFR1 and/or TNFR2 (TNFR1KO, TNFR2KO, and TNFR1/R2KO MSCs). After anesthesia was initiated via inhalation of isoflurane, myocardial ischemia was induced in rats via coronary artery ligation. Hearts were then injected with vehicle or MSCs (1 x 10 cells/mL). Myocardial function was assessed 28 days postsurgery with 2-dimensional echocardiograms and isolated heart perfusion. Myocardial tissue was collected for cytokine analysis and infarct measurements. We found that MSC treatment offered significant protection against myocardial ischemia, namely by decreasing infarct size, improving heart function, and decreasing ventricular remodeling compared with vehicle. Compared with WT-MSCs, TNFR1KO MSCs conferred increased cardiac protection, although TNFR2KO and TNFR1/R2KO MSCs conferred less cardiac protection. In addition, treatment with TNFR1KO MSCs was associated with decreased levels of proinflammatory cytokines and an increased level of vascular endothelial growth factor in the myocardium, whereas treatment with TNFR2KO or TNFR1/R2KO MSCs was associated with increased levels of proinflammatory cytokines and a decreased level of vascular endothelial growth factor compared with treatment with WT-MSCs. We conclude that MSC TNFR1 and TNFR2 play important roles in MSC-mediated cardiac protection after myocardial ischemia.

Mesenchymal stem cells (MSCs) may improve myocardial function after I/R injury via paracrine effects, including the release of growth factors. Genetic modification of MSCs is an appealing method to enhance MSC paracrine action. Ablation of TNF receptor 1 (TNFR1), but not TNFR2, increases MSC growth factor production. In this study, therefore, we hypothesized that 1) preischemic infusion of MSCs derived from TNFR1 knockout (TNFR1KO) mice will further improve myocardial functional recovery and that 2) TNFR2KO and TNFR1/2KO will abolish MSC-mediated protection in the heart after I/R injury. Mesenchymal stem cells were harvested from adult C57BL/6J (wild-type 1 [WT1]), B6129SF2 (WT2), TNFR1KO, TNFR2KO, and TNFR1/2KO mice. Mesenchymal stem cells were cultured and adopted for experiments after passage 3. Isolated hearts from adult male Sprague-Dawley rats were subjected to 25 min of ischemia and 40 min of reperfusion (Langendorff model), during which time myocardial function was continuously monitored. Before ischemia, 1 mL of vehicle or 1 x 10(6) MSCs/mL from WT1, WT2, TNFR1KO, TNFR2KO, or TNFR1/2KO was infused into the hearts (n = 4-6 per group). Treatment of C57BL/6J mice with MSC before ischemia significantly increased cardiac function. TNFR1 knockout MSCs demonstrated greater cardioprotection when compared with WT MSCs after I/R, as exhibited by improved left ventricular developed pressure and +/-dp/dt. However, infusion of MSCs from TNFR2KO and TNFR1/2KO mice either offered no benefit or decreased MSC-mediated cardiac functional recovery in response to I/R when compared with WT MSCs. TNFR1 signaling may damage MSC paracrine effects and decrease MSC-mediated cardioprotection, whereas TNFR2 likely mediates beneficial effects in MSCs.

Mesenchymal stem cells (MSCs) are a promising therapy for acute organ ischemia in part due to their paracrine production of growth factors. However, transplanted cells encounter an inflammatory environment that mitigates their function and survival, and treating the cells with exogenous agents during ex vivo expansion before transplantation is one strategy for overcoming this limitation by enhancing paracrine function. We hypothesized that preconditioning bone marrow MSCs with TGF-alpha would 1) increase MSC production of the critical paracrine factor, vascular endothelial growth factor (VEGF), via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism and 2) enhance myocardial functional recovery in a rat model of acute myocardial I/R injury. To study this, bone marrow MSCs were harvested from adult male mice (C57BL/6J) and treated in vitro for 24 h according to the following groups: 1) control, 2) TGF-alpha (250 ng mL (-1)), 3) TNF-alpha (50 ng mL (-1)), 4) TGF-alpha + TNF-alpha, 5) hypoxia, and 6) TGF-alpha + hypoxia. For the isolated heart perfusion experiments, adult male Sprague-Dawley rat hearts were isolated, perfused via the Langendorff model, and subjected to I/R. Vehicle or MSCs with or without TGF-alpha preconditioning were infused immediately before ischemia. Mesenchymal stem cells were also treated with TGF-alpha alone or in combination with a p38 MAPK inhibitor (SB202190). In vitro, TGF-alpha increased MSC VEGF production alone (157.9 +/- 1.11 - 291.0 +/- 3.74 pg 10 (-5); P < 0.05) and, to a greater extent, in combination with TNF-alpha or hypoxia (364.5 +/- 0.868 and 342.0 +/- 7.92 pg 10(-5) cells, respectively; P < 0.05 vs. TGF-alpha alone). Postischemic myocardial functional recovery was greater in hearts infused with TGF-alpha-preconditioned MSCs compared with untreated MSCs or vehicle. Myocardial IL-1beta and TNF-alpha production and activation of caspase 3 were significantly decreased after infusion of both cell groups. p38 MAPK inhibition suppressed TGF-alpha-stimulated MSC VEGF production and postischemic myocardial recovery. These results suggest that TGF-alpha stimulates MSC VEGF production in part via a p38 MAPK-dependent mechanism, and preconditioning MSCs with TGF-alpha may enhance their ability to protect myocardium during I/R injury.

Multiple studies of left ventricular dysfunction suggest that females may be more resistant to ischemia or endotoxemia. However, sex differences in right ventricular (RV) responses to pressure overload and/or endotoxemia have not been elucidated. We hypothesized that females would maintain better RV function during acute pressure overload (APO), endotoxemia, or a simultaneous insult from both processes. Age-matched male and proestrus female Sprague-Dawley rats were given an intraperitoneal injection of either phosphate buffered saline or LPS. Six hours after injection, hearts were removed by median sternotomy and isolated via Langendorff. End-diastolic pressures were sequentially elevated past physiologic levels by increasing the volume of a latex balloon that was inserted into the RV. Male RV function was depressed to a greater degree after APO injury compared with that in females (developed pressure: male, 44.97 mmHg vs. female, 58.23 mmHg). Interestingly though, at a physiologic end-diastolic pressure of 5 mmHg, endotoxic males and females maintained equivalent RV function. However, with concurrent endotoxic insult and APO, RV function was better maintained in males as compared with that in females (developed pressure: male, 59% of control versus female, 41% of control). Furthermore, tissue levels of IL-1 and IL-6, but not IL-10, were increased after endotoxin exposure but did not differ based on sex. Through this study, we have shown that sex differences exist in RV dysfunction, and that different cardiac insults diversely affect myocardial function. Understanding these differences may allow for the implementation of novel therapeutic treatment options that are designed to attenuate RV cardiovascular collapse.