Brent R. Weil

Preconditioning mesenchymal stem cells with transforming growth factor-alpha improves mesenchymal stem cell-mediated cardioprotection.

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-&agr; 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-&agr; (250 ng mL−1), 3) TNF-&agr; (50 ng mL−1), 4) TGF-&agr; + TNF-&agr;, 5) hypoxia, and 6) TGF-&agr; + 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-&agr; preconditioning were infused immediately before ischemia. Mesenchymal stem cells were also treated with TGF-&agr; alone or in combination with a p38 MAPK inhibitor (SB202190). In vitro, TGF-&agr; 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-&agr; or hypoxia (364.5 ± 0.868 and 342.0 ± 7.92 pg 10−5 cells, respectively; P < 0.05 vs. TGF-&agr; alone). Postischemic myocardial functional recovery was greater in hearts infused with TGF-&agr;-preconditioned MSCs compared with untreated MSCs or vehicle. Myocardial IL-1&bgr; and TNF-&agr; production and activation of caspase 3 were significantly decreased after infusion of both cell groups. p38 MAPK inhibition suppressed TGF-&agr;-stimulated MSC VEGF production and postischemic myocardial recovery. These results suggest that TGF-&agr; stimulates MSC VEGF production in part via a p38 MAPK-dependent mechanism, and preconditioning MSCs with TGF-&agr; may enhance their ability to protect myocardium during I/R injury.

Estrogen receptor β mediates increased activation of PI3K/Akt signaling and improved myocardial function in female hearts following acute ischemia

Females have a lower incidence of heart failure and improved survival after myocardial ischemia-reperfusion (I/R) compared with males. Although estrogen-suppressed cardiomyocyte apoptosis may be mediated through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, it is unclear whether this action is mediated via estrogen receptor beta (ERbeta). Therefore, we hypothesized that ERbeta mediates estrogen-induced cardioprotection through PI3K/Akt and antiapoptotic signaling in females but not in males. Isolated male and female hearts from ERbeta knockout (ERbetaKO) and wild-type (WT) mice (n = 5 mice/group) were subjected to 20-min ischemia followed by 60-min reperfusion (Langendorff). Ablation of ERbeta significantly decreased postischemic recovery of left ventricular developed pressure in female, but not male, hearts. Reduced activation of PI3K and Akt was noted in female ERbetaKO hearts, which was associated with increased expression of caspase-3 and -8, as well as decreased Bcl-2 levels compared with WT. However, myocardial STAT3, SOCS3 (suppressor of cytokine signaling 3), VEGF, and TNF receptors 1 and 2 levels did not change in ERbetaKO of either sex following I/R. Furthermore, deficiency of ERbeta increased myocardial JNK activation in females but increased ERK1/2 activity in males during acute I/R. We conclude that ERbeta mediates myocardial protection via upregulation of PI3K/Akt activation, decreased caspase-3 and -8, and increased Bcl-2 in female hearts following I/R. These findings provide evidence of ERbeta-mediated PI3K/Akt and antiapoptotic signaling in the myocardium and may lend insight into the mechanistic pathways behind the observed variation in clinical outcomes between males and females after myocardial infarction.

Signaling via GPR30 protects the myocardium from ischemia/ reperfusion injury

BACKGROUND Estrogen may protect against the development of cardiovascular disease. Recently, a receptor known as GPR30 that seems to mediate estrogens nongenomic effects has been identified. We hypothesized that the activation of GPR30 protects cardiac function and decreases myocardial inflammation after global ischemia/reperfusion (I/R). METHODS Hearts from male Sprague-Dawley rats were perfused via Langendorff and treated with either (1) vehicle; (2) 10 nm of the GPR30 agonist, G-1; or (3) 100 nm of G-1; they then were subjected to 25 minutes of ischemia and 40 minutes of reperfusion. Cardiac functional parameters were measured continuously. Ventricular tissue was analyzed for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. RESULTS At end-reperfusion, the left ventricular developed pressure in the 100-nm G-1 group was improved compared with vehicle (26% +/- 12% equilibrium vs 54% +/- 9% equilibrium; P < .05). Similar findings were noted when comparing the 100-nm G-1 group with the vehicle in terms of +dP/dt (53% +/- 12% equilibrium vs 26% +/- 19%, respectively; P < .05) and -dP/dt (56% +/- 15% equilibrium vs 22% +/- 16% equilibrium, respectively; P < .05). TNF-alpha, IL-1beta, and IL-6 levels were lower in myocardium of the 100-nm G-1 group compared with the vehicle (P < .05). CONCLUSION The GPR30 agonist, G-1, improves functional recovery and decreases myocardial inflammation after global I/R. GPR30 may play an important role in estrogens ability to protect the heart against I/R injury.

The effects of estrogen on pulmonary artery vasoreactivity and hypoxic pulmonary vasoconstriction: Potential new clinical implications for an old hormone

Background and Objectives:Recent research recognizes gender as a major factor determining the outcomes in trauma, ischemia/reperfusion, shock, and sepsis. In particular, estrogen has been demonstrated to exert protective effects in these settings. The effects of estrogens on the pulmonary vasculature are potent and complex yet not fully understood. A better mechanistic understanding may allow for future therapeutic interventions in pulmonary hypertensive crises after cardiac surgery and during acute lung injury as well as in patients with pulmonary arterial hypertension. Data Sources and Study Selection:We searched PubMed for articles in the English language by using the search words pulmonary hypertension, hypoxic pulmonary vasoconstriction, estrogen, estradiol, inflammation, acute injury, ischemia reperfusion, sepsis, trauma, and burns. These were used in various combinations. We read the abstracts of the relevant titles to confirm their relevance, and the full articles were then extracted. References from extracted articles were checked for any additional relevant articles. Data Extraction and Synthesis:Estrogen plays a critical role in the improved outcomes in the settings of trauma, shock, sepsis, myocardial ischemia/reperfusion, and acute lung injury. Several new mechanisms of action have been identified. In the pulmonary vasculature, estrogen causes vasodilation and attenuates the vasoconstrictor response to various stimuli, including hypoxia. This is mediated by increased levels of prostacyclin and nitric oxide as well as decreased levels of endothelin-1. In addition, effects on intracellular signaling pathways and several kinases as well as anti-inflammatory mechanisms may contribute as well. Recent studies suggest the importance of acute, nongenomic effects. Conclusion:Estrogen exerts a variety of nongenomic actions, which may allow for future therapeutic interventions in pulmonary vascular disease.

Mesenchymal stem cells attenuate myocardial functional depression and reduce systemic and myocardial inflammation during endotoxemia.

BACKGROUND Endotoxemia is associated with depressed cardiac function during sepsis. Mesenchymal stem cells (MSCs) possess an ability to modulate the inflammatory response during sepsis, but it is unknown whether MSCs possess the ability to reduce endotoxemia-induced myocardial injury and dysfunction. METHODS Endotoxemia was induced in rats via injection of lipopolysaccharide (LPS). Animals were divided into the following groups: (1) saline + saline; (2) LPS + saline; (3) LPS + MSCs; and (4) LPS + LLC-PK1 renal epithelial cells (differentiated control). At 6 hours, animals were anesthetized, serum was collected, and hearts were extracted and perfused via the isolated heart system. Hearts and serum were analyzed for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-10. RESULTS The administration of LPS depressed myocardial function. Treatment with MSCs ameliorated this depression. Serum TNF-alpha, IL-1beta, and IL-6 were elevated in LPS-treated groups. Treatment with MSCs was associated with reduced levels of these cytokines. A trend toward reduced myocardial TNF-alpha and significant reductions in myocardial IL-1beta and IL-6 were observed in the MSC-treated group. IL-10 levels were increased after the LPS administration in both serum and myocardium. Serum levels were increased further after treatment with MSCs. CONCLUSION Treatment with MSCs during endotoxemia reduces systemic and myocardial inflammation and is associated with a reduction in LPS-induced myocardial functional depression.

Proinflammatory Cytokine Effects on Mesenchymal Stem Cell Therapy for the Ischemic Heart

Mesenchymal stem cells (MSCs) hold great promise for improving myocardial recovery after ischemia. The cardiothoracic surgeon is uniquely positioned to be at the forefront of any clinical application of this therapy. As such, a basic understanding of stem cells and the cytokines that affect stem cell function will be an essential component of the surgeons ever-expanding knowledge base. This review provides: (1) a general overview of stem cells and MSCs in particular, (2) critically analyzes several cytokines known to alter MSC function, and (3) discusses methods to manipulate cytokine-activated MSCs to improve MSC function for potential clinical application.

Animal Models of Myocardial and Vascular Injury

Over the past century, numerous animal models have been developed in an attempt to understand myocardial and vascular injury. However, the successful translation of results observed in animals to human therapy remains low. To understand this problem, we present several animal models of cardiac and vascular injury that are of particular relevance to the cardiac or vascular surgeon. We also explore the potential clinical implications and limitations of each model with respect to the human disease state. Our results underscore the concept that animal research requires an in-depth understanding of the model, animal physiology, and the potential confounding factors. Future outcome analyses with standardized animal models may improve translation of animal research from the bench to the bedside.

Exogenous estrogen rapidly attenuates pulmonary artery vasoreactivity and acute hypoxic pulmonary vasoconstriction.

Chronic estrogen exposure has been shown to affect pulmonary artery (PA) vasoreactivity. However, the immediate effects of exogenously administered 17&bgr;-estradiol (E2) on vasopressor-induced PA vasoconstriction and acute hypoxic pulmonary vasoconstriction (HPV) have not yet been investigated. We hypothesized that exogenously administered E2 attenuates PA vasoreactivity and acute HPV through a rapid mechanism. Isometric force displacement was measured in isolated PA rings from proestrus female adult Sprague-Dawley rats, estrus, metestrus, or diestrus female adult Sprague-Dawley rats, and male adult Sprague-Dawley rats. The vasoconstrictor response in the absence of hypoxia (organ bath bubbled with 95% O2/5% CO2) was measured after stimulation with 1 &mgr;M of phenylephrine. Hypoxia was generated by changing the gas to 95% N2/5% CO2. The E2 was added to the organ bath in 0.1-nM, 0.5-nM, 1-&mgr;M, 500-&mgr;M, and 1-mM doses. The 1-mM dose caused an immediate decrease in force in PA rings from estrus, metestrus, or diestrus female adult Sprague-Dawley rats. In addition, 500 &mgr;M and 1 mM of E2 attenuated phenylephrine- and hypoxia-induced vasoconstriction and potentiated the vasodilatory phase of hypoxia. These effects were immediate and independent of sex or estrous cycle. Lower E2 doses did not mediate any significant effects. We conclude that high doses of exogenous E2 acutely attenuate PA vasoreactivity and acute HPV in a rapid and dose-dependent manner. A better understanding of how E2 modulates the pulmonary vasomotor response may allow for future therapeutic interventions in acute pulmonary hypertensive crises or in pulmonary arterial hypertension.

Cell-Based Therapy for Ischemic Heart Disease: A Clinical Update

Progenitor cell therapy is a promising treatment for ischemic heart disease. Early clinical trials of autologous bone marrow-derived progenitor cell therapy for acute and chronic myocardial ischemia showed modest functional improvements after cell delivery; however, the duration of these benefits remains unclear. Ongoing investigations continue to enhance our understanding of the mechanisms by which progenitor and stem cells function and how their survival and cardioprotective abilities can be improved. This review discusses: (1) relevant progenitor and stem cells in myocardial regenerative therapy, (2) routes of cell delivery to ischemic myocardium, (3) clinical trials investigating bone marrow-derived progenitor cell therapy for myocardial ischemia, and (4) future directions of the field.

TNF receptor 2, not TNF receptor 1, enhances mesenchymal stem cell-mediated cardiac protection following acute 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 × 106 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.ABBREVIATIONS-MSC-bone marrow mesenchymal stem cell; TNFR1-TNF receptor 1; TNFR2-TNF receptor 2; TNFR1KO-TNFR1 knockout; TNFR2KO-TNFR2 knockout; TNFR1/2KO-TNFR1 and TNFR2 knockout; WT1-C57BL/6J; WT2-B6129SF2; LVDP-left ventricular developed pressure; EDP-end-diastolic pressure; VEGF-vascular endothelial growth factor