Jeremy L. Herrmann

VEGF is critical for stem cell-mediated cardioprotection and a crucial paracrine factor for defining the age threshold in adult and neonatal stem cell function

Bone marrow mesenchymal stem cells (MSCs) may be a novel treatment modality for organ ischemia, possibly through the release of beneficial paracrine factors. However, an age threshold likely exists as to when MSCs gain their beneficial protective properties. We hypothesized that 1) VEGF would be a crucial stem cell paracrine mediator in providing postischemic myocardial protection and 2) small-interfering (si)RNA ablation of VEGF in adult MSCs (aMSCs) would equalize the differences observed between aMSC- and neonatal stem cell (nMSC)-mediated cardioprotection. Female adult Sprague-Dawley rat hearts were subjected to ischemia-reperfusion injury via Langendorff-isolated heart preparation (15 min equilibration, 25 min ischemia, and 60 min reperfusion). MSCs were harvested from adult and 2.5-wk-old neonatal mice and cultured under normal conditions. VEGF was knocked down in both cell lines by VEGF siRNA. Immediately before ischemia, one million aMSCs or nMSCs with or without VEGF knockdown were infused into the coronary circulation. The cardiac functional parameters were recorded. VEGF in cell supernatants was measured via ELISA. aMSCs produced significantly more VEGF than nMSCs and were noted to increase postischemic myocardial recovery compared with nMSCs. The knockdown of VEGF significantly decreased VEGF production in both cell lines, and the pretreatment of these cells impaired stem cell-mediated myocardial function. The knockdown of VEGF in adult stem cells equalized the myocardial functional differences observed between adult and neonatal stem cells. Therefore, VEGF is a critical paracrine mediator in facilitating postischemic myocardial recovery and likely plays a role in mediating the observed age threshold during stem cell therapy.

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.

Stem cell mechanisms and paracrine effects: potential in cardiac surgery.

Heart disease remains the leading cause of death in the industrialized world. Stem cell therapy is a promising treatment modality for injured cardiac tissue. A novel mechanism for this cardioprotection may include paracrine actions. Cardiac surgery represents the unique situation where preischemia and postischemia treatment modalities exist that may use stem cell paracrine protection. This review (1) recalls the history of stem cells in cardiac disease and the unraveling of its mechanistic basis for protection, (2) outlines the pathways for stem cell-mediated paracrine protection, (3) highlights the signaling factors expressed, (4) explores the potential of using stem cells clinically in cardiac surgery, and (5) summarizes all human stem cell studies in cardiac disease to date.ABBREVIATIONS-MSC- mesenchymal stem cell; VEGF- vascular endothelial growth factor; HGF- hepatocyte growth factor; FGF- fibroblast growth factor; TGF- transforming growth factor; LV- left ventricular; LVEF- left ventricular ejection fraction; MI- myocardial infarction; PCI- percutaneous coronary intervention

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.

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.

Testosterone-Down-Regulated Akt Pathway During Cardiac Ischemia/ Reperfusion: A Mechanism Involving BAD, Bcl-2 and FOXO3a

BACKGROUND Lower levels of myocardial Akt activity in males are associated with a higher incidence of heart failure and worsened cardiac function after ischemia/reperfusion (I/R). While Akt activation by estrogen provides cardioprotection in females, no information exists regarding the effect of testosterone on the myocardial Akt pathway following I/R. We hypothesized that following I/R: (1) endogenous testosterone will decrease myocardial Akt activation in male hearts; (2) endogenous testosterone will mediate downstream signals of Akt, including Bad, Bcl-2, and FOXO3a; (3) administration of exogenous testosterone will recapitulate negative effects on the Akt pathway in castrated male hearts. METHODS AND RESULTS Rat hearts from age-matched adult males, females, castrated males, males with androgen receptor blocker-flutamide, castrated males with chronic 5α-dihydrotestosterone (DHT) implantation, or acute testosterone infusion (ATI) (n = 9/group) were subjected to I/R (Langendorff). Castration or flutamide treatment significantly up-regulated myocardial Akt activation, increased downstream apoptosis-regulatory molecules p-Bad, Bcl-2, p-FOXO3a, but reduced Fas-L, consistent with decreased myocardial injury in male hearts following I/R. ATI administration, but not chronic DHT, reversed these effects on Akt signaling associated with further exacerbated cardiac dysfunction in castrated males. Notably, lower levels of MnSOD were observed in male hearts, and castration or flutamide treatment restored myocardial MnSOD expression to the levels of females in male hearts after I/R. CONCLUSION Our study represents the initial evidence of testosterone-induced down-regulation of the Akt pathway in male hearts following I/R, thereby mediating cardiac injury through decreased p-Bad, reduced ratio of Bcl-2/Bax in the cytoplasm, and increased FOXO3a in the nucleus.

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