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Background Neonatal mesenchymal stem cells exhibit less cardioprotective potential than their adult counterparts. Transforming growth factor-α (TGF-α) has been shown to stimulate adult stem cell VEGF production, however, it remains unknown whether it may augment neonatal stem cell paracrine function. We hypothesized that TGF-α would equalize adult and neonatal stem cell paracrine function and cardioprotection during acute ischemia/reperfusion. Materials and Methods Bone marrow mesenchymal stem cells isolated from adult and 2.5 wk-old mice were treated with TGF-α (250 ng/mL) for 24 h. VEGF, HGF, IGF-1, IL-1β, and IL-6 production were measure in vitro , and cells were infused via an intracoronary route using a model of isolated heart perfusion. Results TGF-α equalized adult and neonatal stem cell VEGF production but did not affect production of HGF, IGF-1, IL-1β, or IL-6. ERK, p38 MAPK, and JNK phosphorylation were greater in adult cells in response to TGF-α. Whereas infusion of adult but not neonatal stem cells was associated with improved myocardial functional recovery during reperfusion, infusions of either TGF-α-pretreated cell group were associated with the greatest functional recovery. TGF-α equalizes adult and neonatal mesenchymal stem cell VEGF production and cardioprotection in association with differential regulation of ERK, p38 MAPK, and JNK phosphorylation.

Background Mesenchymal stem cells (MSCs) improve postischemic myocardial function in part through their secretion of growth factors such as vascular endothelial growth factor (VEGF). Pretreating MSCs with various cytokines or small molecules can improve VEGF secretion and MSC-mediated cardioprotection. However, whether 1 cytokine can potentiate the effect of another cytokine in MSC pretreatment to achieve a synergistic effect on VEGF production and cardioprotection is poorly studied. Methods MSCs were treated with interleukin (IL)-1β and/or transforming growth factor (TGF)-β1 for 24 hours before experiments. VEGF production was determined by enzyme-linked immunosorbent assay. Isolated hearts from adult male Sprague-Dawley rats were subjected to 15 minutes of equilibration, 25 minutes of ischemia, and 40 minutes reperfusion. Hearts ( n = 5–7 per group) were randomly infused with vehicle, untreated MSCs, or MSCs pretreated with IL-1β and/or TGF-β1. Specific inhibitors were used to delineate the roles of p38 mitogen-activated protein kinase (MAPK) and SMAD3 in IL-1β– and TGF-β1–mediated stimulation of MSCs. Results MSCs cotreated with IL-1β and TGF-β1 exhibited synergistically increased VEGF secretion, and they greatly improved postischemic myocardial functional recovery. Ablation of p38 MAPK and SMAD3 activation with specific inhibitors negated both IL-1β– and TGF-β1–mediated VEGF production in MSCs and the ability of these pretreated MSCs to improve myocardial recovery after ischemia. Conclusion Pretreating MSCs with 2 cytokines may be useful to fully realize the potential of cell-based therapies for ischemic tissues.

Background Sepsis remains a source of morbidity and mortality in the postoperative patient despite appropriate resuscitative and antimicrobial approaches. Recent research has focused upon additional interventions such as exogenous cell-based therapy. Mesenchymal stem cells (MSCs) exhibit multiple beneficial properties through their capacity for homing, attenuating the inflammatory response, modulating immune cells, and promoting tissue healing. Recent animal trials have provided evidence that MSCs may be useful therapeutic adjuncts. Materials and Methods A directed search of recent medical literature was performed utilizing PubMed to examine the pathophysiology of sepsis, mechanisms of mesenchymal stem cell interaction with host cells, sepsis animal models, and recent trials utilizing stem cells in sepsis. Results MSCs continue to show promise in the treatment of sepsis by their intrinsic ability to home to injured tissue, secrete paracrine signals to limit systemic and local inflammation, decrease apoptosis in threatened tissues, stimulate neoangiogenesis, activate resident stem cells, beneficially modulate immune cells, and exhibit direct antimicrobial activity. These effects are associated with reduced organ dysfunction and improved survival in animal models. Conclusion Research utilizing animal models of sepsis has provided a greater understanding of the beneficial properties of MSCs. Their capacity to home to sites of injury and use paracrine mechanisms to change the local environment to ultimately improve organ function and survival make MSCs attractive in the treatment of sepsis. Future studies are needed to further evaluate the complex interactions between MSCs and host tissues.

Background Cardiac surgery induces the release of inflammatory mediators that can prolong cardiac dysfunction after operative intervention. Interleukin-10 (IL-10), a potent inhibitor of myocardial inflammation, is a known factor in myocardial protection after ischemia/reperfusion (I/R) injury. We hypothesized that IL-10 activity during initial reperfusion is mediated through the signal transducer and activator of transcription 3 (STAT3) pathway. Methods Adult rat hearts were isolated and perfused via Langendorff protocol and subjected to global I/R. After determining the effective IL-10 dose, hearts were administered vehicle, IL-10, or IL-10 + Stattic (specific STAT3 inhibitor) 1 min prior to ischemia. After reperfusion, hearts were sectioned and assessed for levels of myocardial inflammatory cytokines and protein. Results The IL-10 minimum effective dose was 1 μg. IL-10-treated hearts had improved markedly myocardial function after global I/R compared to both vehicle and IL-10 + Stattic groups. In addition, IL-10 treatment was associated with a significant decrease in myocardial interleukin-1β (IL-1β) and interleukin-6 (IL-6) and increase in myocardial IL-10. Myocardial STAT3 was elevated markedly in IL-10 treated hearts. Conclusion IL-10 improves myocardial function after acute global I/R and suppresses inflammation through the STAT3 pathway. The administration of anti-inflammatory agents may have potential therapeutic applications in cardiac surgery.

Background Mesenchymal stem cells (MSCs) hold promise for the treatment of renal disease. While MSCs have been shown to accelerate recovery and prevent acute renal failure in multiple disease models, the effect of MSC therapy on chronic obstruction-induced renal fibrosis has not previously been evaluated. Materials and Methods Male Sprague-Dawley rats underwent renal artery injection of vehicle or fluorescent-labeled human bone marrow-derived MSCs immediately prior to sham operation or induction of left ureteral obstruction (UUO). One or 4 wk later, the kidneys were harvested and the renal cortex analyzed for evidence of stem cell infiltration, epithelial-mesenchymal transition (EMT) as evidenced by E-cadherin/α-smooth muscle actin (α-SMA) expression and fibroblast specific protein (FSP+) staining, renal fibrosis (collagen content, Masson's trichrome staining), and cytokine and growth factor activity (ELISA and real time RT-PCR). Results Fluorescent-labeled MSCs were detected in the interstitium of the kidney up to 4 wk post-obstruction. Arterially delivered MSCs significantly reduced obstruction-induced α-SMA expression, FSP+ cell accumulation, total collagen content, and tubulointerstitial fibrosis, while simultaneously preserving E-cadherin expression, suggesting that MSCs prevent obstruction-induced EMT and renal fibrosis. Exogenous MSCs reduced obstruction-induced tumor necrosis factor-α (TNF-α) levels, but did not alter transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), fibroblast growth factor (FGF), or hepatocyte growth factor (HGF) expression. Conclusions Human bone marrow-derived MSCs remain viable several weeks after delivery into the kidney and provide protection against obstruction-induced EMT and chronic renal fibrosis. While the mechanism of MSCs-induced renal protection during obstruction remains unclear, our results demonstrate that alterations in TNF-α production may be involved.

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.

Mesenchymal stem cells (MSCs) have been used experimentally and clinically in the treatment of a wide variety of pathologies. It is now clear that a number of different mechanisms contribute to the therapeutic effects exerted by these cells. The ability of MSCs to interact with and modulate the functions of a wide variety of immune cells has been recognized as one such mechanism. The implications that the immunomodulatory properties of MSCs may have for the treatment of solid organ rejection, the Systemic Inflammatory Response Syndrome, cancer, and Crohn’s disease are reviewed herein.

Background Stem cell-based therapies for myocardial ischemia have demonstrated promising early clinical results, but their benefits have been limited in duration due to impaired donor cell engraftment and function. Several strategies have emerged for enhancing stem cell function prior to their therapeutic use particularly with regard to stem cell homing, paracrine function, and survival. This review discusses current understandings of stem cell-mediated cardioprotection as well as methods of enhancing post-transplantation stem cell function and survival through hypoxic preconditioning, genetic manipulation, and pharmacologic pretreatment. Materials and Methods A literature search was performed using the MEDLINE and PubMed databases using the keywords “stem cell therapy,” “myocardial ischemia,” “hypoxic preconditioning,” “paracrine function,” and “stem cell pretreatment.” Studies published in English since January 1990 were selected. In addition, studies were identified from references cited in publications found using the search terms. Results All included studies utilized animal studies and/or in vitro techniques. Stem cell modifications generally targeted stem cell homing (SDF-1, CXCR4), paracrine function (VEGF, angiogenin, Ang-1, HGF, IL-18 binding protein, TNFR1/2), or survival (Akt, Bcl-2, Hsp20, HO-1, FGF-2). However, individual modifications commonly exhibited pleiotropic effects involving some or all of these general categories. Conclusion These strategies for optimizing stem cell-mediated cardioprotection present unique potential sets of advantages and disadvantages for clinical application. Additional questions remain including those that are most efficacious in terms of magnitude and duration of benefit as well as whether combinations may yield greater benefits in both the preclinical and clinical settings.

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.

Atrial fibrillation (AF) is the most common chronic arrhythmia in the United States and is associated with high morbidity rates and significant healthcare costs. Although medical therapy for AF modestly reduces complications, cardiac surgery continues to have an important role in the treatment of AF and is constantly evolving. Cardiothoracic surgeons are uniquely positioned to offer effective operations to patients with lone AF, in addition to those undergoing concomitant elective cardiac surgery. This review discusses (1) the burden of AF, (2) classification and electrophysiology of AF, (3) surgical techniques and outcomes, and (4) future directions in surgical therapy.

Acute right ventricular (RV) failure is a frequent and serious clinical challenge in the intensive care unit. It is usually seen as a consequence of left ventricular failure, pulmonary embolism, pulmonary hypertension, sepsis, acute lung injury or after cardiothoracic surgery. The presence of acute RV failure not only carries substantial morbidity and mortality, but also complicates the use of commonly used treatment strategies in critically ill patients. In contrast to the left ventricle, the RV remains relatively understudied, and investigations of the treatment of isolated RV failure are rare and usually limited to nonrandomized observations. We searched PubMed for papers in the English language by using the search words right ventricle, right ventricular failure, pulmonary hypertension, sepsis, shock, acute lung injury, cardiothoracic surgery, mechanical ventilation, vasopressors, inotropes, and pulmonary vasodilators. These were used in various combinations. We read the abstracts of the relevant titles to confirm their relevance, and the full papers were then extracted. References from extracted papers were checked for any additional relevant papers. This review summarizes the general measures, ventilation strategies, vasoactive substances, and surgical as well as mechanical approaches that are currently used or actively investigated in the treatment of the acutely failing RV.

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)-α, interleukin (IL)-1β, IL-6, and IL-10. Results The administration of LPS depressed myocardial function. Treatment with MSCs ameliorated this depression. Serum TNF-α, IL-1β, 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-α and significant reductions in myocardial IL-1β 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.

Estrogen may protect against the development of cardiovascular disease. Recently, a receptor known as GPR30 that seems to mediate estrogen's 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).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.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).The GPR30 agonist, G-1, improves functional recovery and decreases myocardial inflammation after global I/R. GPR30 may play an important role in estrogen's ability to protect the heart against I/R injury.

The potential use of stem cells for acute and chronic renal injury is under intensive investigation. We summarized the current literature on the potential therapeutic role of mesenchymal stem cells for kidney injury.We reviewed the pertinent literature on mesenchymal stem cell therapy for acute and chronic renal injury.Experimental evidence suggests that administering exogenous mesenchymal stem cells during acute and chronic kidney injury may improve functional and structural recovery of the tubular, glomerular and interstitial kidney compartments. Several studies point to a paracrine and/or endocrine mechanism of action rather than to direct repopulation of cells in the injured nephron. Multiple questions remain unanswered regarding the protective action of mesenchymal stem cells during renal injury, including signals that regulate stem cell homing to injured tissue, factors regulating paracrine and/or endocrine activity of exogenous mesenchymal stem cells and particularly the long-term behavior of administered stem cells in vivo.Many questions remain unanswered but mesenchymal stem cell based therapy is a promising new strategy for acute and chronic kidney disease.

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.

Toll-like receptor 2 (TLR2), a key component of the innate immune system, is linked to inflammation and myocardial dysfunction after ischemia-reperfusion injury (I/R). Treatment of the heart with mesenchymal stem cells (MSCs) is known to improve myocardial recovery after I/R in part by paracrine factors such as VEGF. However, it is unknown whether TLR2 activation on the MSCs affects MSC-mediated myocardial recovery and VEGF production. We hypothesized that the knockout of TLR2 on the MSCs (TLR2KO MSCs) would 1) improve MSC-mediated myocardial recovery and 2) increase myocardial and MSC VEGF release. With the isolated heart perfusion system, Sprague-Dawley rat hearts were subjected to I/R and received one of three intracoronary treatments: vehicle, male wild-type MSCs (MWT MSCs), or TL2KO MSCs. All treatments were performed immediately before ischemia, and heart function was measured continuously. Postreperfusion, heart homogenates were analyzed for myocardial VEGF production. Contrary to our hypothesis, only MWT MSC treatment significantly improved the recovery of left ventricular developed pressure and the maximal positive and negative values of the first derivative of pressure. In addition, VEGF production was greatest in hearts treated with MWT MSCs. To investigate MSC production of VEGF, MSCs were activated with TNF in vitro and the supernatants collected for ELISA. In vitro basal levels of MSC VEGF production were similar. However, with TNF activation, MWT MSCs produced significantly more VEGF, whereas activated TLR2KO MSC production of VEGF was unchanged. Finally, we observed that MWT MSCs proliferated more rapidly than TLR2KO MSCs. These data indicate that TLR2 may be essential to MSC-mediated myocardial recovery and VEGF production.

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.

IL-18 is a proinflammatory cytokine known to cause tissue injury by inducing inflammation and cell death. Increased levels of IL-18 are associated with myocardial injury after ischemia or infarction. IL-18-binding protein (IL-18BP), the naturally occurring inhibitor of IL-18 activity, decreases the severity of inflammation in response to injury. In the present study, mesenchymal stem cells (MSCs) derived from mice transgenic for over expression of human IL-18BP were tested in rat models of global myocardial ischemia and acute myocardial infarction. Improved myocardial function is associated with production of VEGF, and in vitro, IL-18BP MSCs secreted higher levels of constitutive VEGF compared to wild-type MSCs. Whereas IL-18 increased cell death and reduced VEGF in wild-type MSCs, IL-18BP MSCs were protected. In an isolated heart model, intracoronary infusion of IL-18BP MSCs before ischemia increased postischemic left ventricular (LV) developed pressure to 79.5 + or - 9.47 mmHg compared to 59.3 + or - 7.8 mmHg in wild-type MSCs and 37.8 + or - 5 mmHg in the vehicle group. Similarly, using a coronary artery ligation model, intramyocardial injection of IL-18BP MSCs improved LV ejection fraction to 67.8 + or - 1.76% versus wild-type MSCs (57.4 + or - 1.33%) and vehicle (39.2 + or - 2.07%), increased LV fractional shortening 1.25-fold over wild-type MSCs and 1.95-fold over vehicle, decreased infarct size to 38.8 + or - 2.16% compared to 46.4 + or - 1.92% in wild-type MSCs and 60.7 + or - 2.2% in vehicle, reduced adverse ventricular remodeling, increased myocardial VEGF production, and decreased IL-6 levels. This study provides the concept that IL-18BP genetically modified stem cells improve cardioprotection over that observed with unmodified stem cells.

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 surgeon's 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.

Optimizing the function and proliferative capacity of stem cells is essential to maximize their therapeutic benefits. High glucose concentrations are known to have detrimental effects on many cell types. We hypothesized that human mesenchymal stem cells (hMSCs) cultured in high glucose-containing media would exhibit diminished proliferation and attenuated production of VEGF, hepatocyte growth factor (HGF), and FGF2 in response to treatment with TNF-α, LPS, or hypoxia. hMSCs were plated in medium containing low (5.5 mM) and high (20 mM or 30 mM) glucose concentrations and treated with TNF-α, LPS, or hypoxia. Supernatants were collected at 24 and 48 h and assayed via ELISA for VEGF, HGF, and FGF2. In addition, hMSCs were cultured on 96-well plates at the above glucose concentrations, and proliferation at 48 h was determined via bromo-2′-deoxy-uridine (BrdU) incorporation. At 24 and 48 h, TNF-α, LPS, and hypoxia-treated hMSCs produced significantly higher VEGF, HGF, and FGF2 compared with control. Hypoxia-induced VEGF production by hMSCs was the most pronounced change over baseline. At both 24 and 48 h, glucose concentration did not affect production of VEGF, HGF, or FGF2 by untreated hMSCs and those treated with TNF-α, LPS, or hypoxia. Proliferation of hMSCs as determined via BrdU incorporation was unaffected by glucose concentration of the media. Contrary to what has been observed with other cells, hMSCs may be resistant to the short-term effects of high glucose. Ongoing efforts to characterize and optimize ex vivo and in vivo conditions are critical if the therapeutic benefits of MSCs are to be maximized.

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.

Angiogenic factors have been the focus of a great deal of research for the treatment of ischemic diseases. Described just more than 10 years ago, angiopoietin 1 (Ang-1) has shown promising results in I/R models. Angiopoietin 1 is essential for both embryonic vasculogenesis and adult angiogenesis. Because of its role in maturation of vessels, it seems well suited to complement the angiogenic factor vascular endothelial growth factor (VEGF), which has been shown to induce vascular budding but in isolation produces nonfunctional vessels. This review will focus on (1) Ang-1 and its receptor, Tie-2, and the resultant intracellular signaling cascade; (2) the complex relationship of Ang-1 and VEGF; (3) the results of Ang-1 in I/R and sepsis models; (4) the results of combination (Ang-1 and VEGF) therapies in I/R and sepsis models; and (5) delivery mechanisms for angiogenic factors to ischemic heart.

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-α or ER-β, and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-α agonist propylpyrazole triol (PPT) and/or the selective ER-β agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings ( n = 3–10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine (10−8M − 10−5M) and hypoxia (Po2 35–45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine (10−8M − 10−4M) and sodium nitroprusside (10−9M − 10−5M). PPT or DPN (10−9M − 5 × 10−5M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) (10−4M). Selective ER-α activation (PPT, 5 × 10−5M) rapidly (−5M) rapidly decreased phase II of hypoxic pulmonary vasoconstriction (HPV). l-NAME eliminated this phenomenon. Lower PPT or DPN concentrations were less effective. We conclude that both ER-α and ER-β decrease PA vasoconstriction. The immediate onset of effect suggests a nongenomic mechanism. The contribution of specific ERs appears to be stimulus specific, with ER-α primarily modulating phenylephrine-induced vasoconstriction, and ER-β inhibiting HPV. NO inhibition eliminates these effects, suggesting a central role for NO in mediating the pulmonary vascular effects of both ER-α and ER-β.

Background Tumor necrosis factor-alpha (TNF-α) plays a critical role in myocardial dysfunction following acute injury. It is unknown, however, if a gender-specific response to TNF infusion exists in isolated rat hearts. Elucidating such mechanisms is important to understanding the myocardial gender differences during acute injury. We hypothesize that females will exhibit a relative resistance to TNF-induced myocardial dysfunction compared to males and that menstrual cycle would influence the degree of female myocardial resistance to TNF-induced myocardial functional depression. Materials and methods Adult male, proestrus female, and metestrus/diestrus female hearts were subjected to 60 min of TNF infusion at 10,000 pg/mL · min via Langendorff. Myocardial contractile function (left ventricular developed pressure, and the positive/negative first derivative of pressure) was continuously recorded. Results 10,000 pg/mL · min of TNF markedly depressed myocardial function in males compared with other doses of TNF. Myocardial function was significantly decreased in males compared to females following TNF infusion. Additionally, both the proestrus and the metestrus/diestrus females exhibited equal resistance to TNF-induced myocardial dysfunction. Conclusion Our study shows that females exhibit a significantly greater degree of resistance to TNF-induced myocardial depression. Moreover, data from this study suggest that fluctuations in estrogen during the reproductive cycle may have little to no influence on TNF-induced myocardial depression.

The production of growth factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by human bone marrow mesenchymal stem cells (MSCs) may play an important role in their paracrine effects on proliferation, differentiation, and protection. NO is produced during ischemia and may affect MSC function. However, it is unknown whether NO alters the production of VEGF and HGF from MSCs. To study this, human MSCs were stimulated to produce growth factors with TNF or LPS with and without various doses of NO donors or NOS inhibitors. We found that FK409, an NO donor, significantly suppressed the production of VEGF and HGF from human MSCs. Vascular endothelial growth factor in the supernatants of cells treated by 20 nM FK409 (497 +/- 19 pg/mL) was significantly lower compared with controls (625 +/- 34 pg/mL). Similarly, NO donor significantly suppressed the amount of HGF from controls (118 +/- 3 to 40 +/- 2 pg/mL) after treatment with 20 nM FK409. NO donor also abolished the augmentation of VEGF production induced by LPS. The amount of VEGF in the supernatant was 571 +/- 11 pg/mL when cells were treated with 20 nM FK409 and LPS (200 ng/mL), which was significantly lower than groups treated with LPS alone (941 +/- 30 pg/mL). This study constitutes an initial report regarding the effect of NO on human MSC growth factor production.

Transforming growth factor-alpha (TGF-alpha) may be an important mediator of wound healing and the injury response. Human bone marrow mesenchymal stem cells (MSCs) release VEGF as a potentially beneficial paracrine response; however, it remains unknown whether TGF-alpha stimulates the production of VEGF from MSCs and, if so, by which mechanisms. We hypothesized that TGF-alpha would increase human MSC VEGF production by MAP kinase kinase (MAPKK/MEK), phosphatidylinositol 3-kinase (PI3-K)-, ERK, and JNK-dependent mechanisms. To study this, MSCs were cultured and divided into the following groups: 1) with vehicle; 2) with various stimulants alone: TGF-alpha, TNF-alpha, or TGF-alpha+TNF-alpha; 3) with individual kinase inhibitors alone (two different inhibitors for each of the following kinases: MEK, PI3-K, ERK, or JNK); and 4) with the above stimulants and each of the eight inhibitors. After 24-h incubation, a TGF-alpha dose-response curve demonstrated that low-dose TGF-alpha (500 pg/ml) suppressed MSC production of VEGF compared with vehicle (502 +/- 16 pg/10(5) cells/ml to 332 +/- 9 pg/10(5) cells/ml), while high-dose TGF-alpha (250 ng/ml) significantly increased MSC VEGF production (603 +/- 24 pg/10(5) cells/ml). High-dose TGF-alpha also increased TNF-alpha-stimulated release of VEGF from MSCs. MSCs exposed to TGF-alpha and/or TNF-alpha also demonstrated increased activation of PI3-K, JNK, and ERK. The TGF-alpha-stimulated production of VEGF by MSCs and the additive effect of TNF-alpha and TGF-alpha on VEGF production were abolished by MEK and PI3-K inhibition, but not ERK or JNK inhibition. Our data suggest that TGF-alpha increases VEGF production in MSCs via MEK- and PI3-K- but not ERK- or JNK-dependent mechanisms.

End-organ ischemia is a common source of patient morbidity and mortality. Stem cell therapy represents a novel treatment modality for ischemic diseases and may aid injured tissues through the release of beneficial paracrine mediators. Female bone marrow mesenchymal stem cells (MSCs) have demonstrated a relative resistance to detrimental TNF receptor 1 (TNFR1) signaling and are thought to be superior to male stem cells in limiting inflammation. However, it is not known whether sex differences exist in TNF receptor 2 (TNFR2)-ablated MSCs. Therefore, we hypothesized that 1) sex differences would be observed in wild-type (WT) and TNFR2-ablated MSC cytokine signaling, and 2) the production of IL-6, VEGF, and IGF-1 in males, but not females, would be mediated through TNFR2. MSCs were harvested from male and female WT and TNFR2 knockout (TNFR2KO) mice and were subsequently exposed to TNF (50 ng/ml) or LPS (100 ng/ml). After 24 h, supernatants were collected and measured for cytokines. TNF and LPS stimulated WT stem cells to produce cytokines, but sex differences were only seen in IL-6 and IGF-1 after TNF stimulation. Ablation of TNFR2 increased VEGF and IGF-1 production in males compared with wild-type, but no difference was observed in females. Female MSCs from TNFR2KOs produced significantly lower levels of VEGF and IGF-1 compared with male TNFR2KOs. The absence of TNFR2 signaling appears to play a greater role in male MSC cytokine production. As a result, male, but not female stem cell cytokine production may be mediated through TNFR2 signaling cascades.

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.

Background Gender differences have been noted in acute ischemia/reperfusion injury. Estrogen and the estrogen receptors (ER) appear to play a critical role in cardiovascular gender differences, given that females have improved myocardial functional recovery associated with decreased tissue inflammation. It has been suggested that ER beta plays a part in decreasing myocardial inflammation following hemorrhage. It remains unknown, however, whether ER beta also may be protective following the more severe insult of complete global, normothermic ischemia/reperfusion injury in the isolated mouse heart. Methods Adult male and female wild-type (WT) and ER beta knockout (ERbKO) mouse hearts were subjected to 20 minutes ischemia and 60 minutes reperfusion (Langendorff model). Myocardial contractile function (±dP/dt) was continuously recorded. Heart tissue was analyzed for tumor necrosis factor, interleukin (IL)-1β, IL-6, and IL-10 levels as determined by enzyme-linked immunosorbent assay. Results Females had markedly improved functional recovery compared with males following ischemia/reperfusion. This recovery was associated with lower levels of myocardial tumor necrosis factor, IL-1β, and IL-6 in females. However, ERbKO females exhibited significantly less postischemic functional recovery than WT females and were similar to WT males. Interestingly, increased myocardial production of tumor necrosis factor, IL-1β, and IL-6 was noted in ERbKO female hearts in response to ischemia/reperfusion. No significant differences were found between male WT and male ERbKO in postischemic functional recovery and proinflammatory cytokine production. Conclusion ER beta plays a role in the protective effects of estrogen following global, warm ischemia/reperfusion of the isolated mouse heart. This understanding ultimately may enable the development of pharmaceutical agents that harness such protection with minimal collateral sex hormone effects.

Stem cells have shown promise for the treatment of end organ ischemia. NFkB has been demonstrated to regulate growth factor secretion in human adult bone marrow stem cells (aBMSCs). We hypothesized that: (1) NFkB is an important mediator in aBMSC and neonatal BMSC (nBMSC) VEGF and IL-6 secretion; and (2) inhibition of NFkB will result in a decrease of VEGF and IL-6 in nBMSCs. BMSCs were plated and exposed to TNF (50 ng/ml) or LPS (100 ng/ml), with or without NFkB or IKK inhibition. VEGF and IL-6 were measured via ELISA in 24-h supernatants. Inhibition of NFkB and IKK both demonstrated a decrease in VEGF (p0.05) in aBMSCs but not nBMSCs. The LPS-stimulated nBMSC with IKK inhibition group was the only exception which demonstrated a decrease in VEGF secretion. However, both NFkB inhibition caused both aBMSCs and nBMSCs to produced less IL-6 after LPS stimulation (p0.05). Only aBMSCs' secretion of IL-6 decreased with NFkB and IKK inhibition when stimulated with TNF (p0.05) differing only when TNF-stimulated nBMSCs were inhibited with IKK. VEGF and IL-6 secretion in aBMSCs is dependent on the classic NFkB pathway. However, neonatal BMSC VEGF and IL-6 secretion is stimulant-specific and utilization of the NFkB pathway is more complex.

Tubulointerstitial fibrosis is the final common pathway to end stage renal disease. The pathophysiology of renal fibrosis involves fibroblast proliferation, macrophage infiltration, the elaboration of cytokines and other proinflammatory mediators, and an imbalance in extracellular matrix deposition and degradation. Although the exact origin of activated fibroblasts remains uncertain, emerging evidence indicates that mature tubular epithelial cells are capable of transforming into myofibroblasts under pathological conditions, a process that is called epithelial-mesenchymal transition.We reviewed the pertinent literature from January 1980 through June 2007 with regard to the contribution of epithelial-mesenchymal transition to renal fibrogenesis.Epithelial-mesenchymal transition is an orchestrated, highly regulated process that proceeds in stepwise fashion and appears to contribute significantly to renal fibrosis and the progression of chronic renal disease. Several cytokines and growth factors regulate epithelial-mesenchymal transition, of which transforming growth factor-beta1 is the most studied.Epithelial-mesenchymal transition is a cellular mechanism that has long been recognized as a central feature of normal development. However, increasing evidence implicates epithelial-mesenchymal transition in the pathophysiology of tubulointerstitial fibrosis and chronic renal disease. Recent insights into the molecular events and intrinsic signaling pathways that are active during epithelial-mesenchymal transition have evoked novel therapeutic strategies aimed at halting the onset and progression of chronic renal fibrosis.

Gender dimorphisms exist in the pathogenesis of a variety of cardiovascular, cardiopulmonary, neurodegenerative, and endocrine disorders. Estrogens exert immense influence on myocardial remodeling following ischemic insult, partially through paracrine growth hormone production by bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells. Estrogens also facilitate the mobilization of endothelial progenitor cells to the ischemic myocardium and enhance neovascularization at the ischemic border zone. Moreover, estrogens limit pathological myocardial remodeling through the inhibitory effects on the proliferation of the cardiac fibroblasts. Androgens also may stimulate endothelial progenitor cell migration from the bone marrow, yet the larger role of androgens in disease pathogenesis is not well characterized. The beneficial effects of sex steroids include alteration of lipid metabolism in preadipocytes, modulation of bone metabolism and skeletal maturation, and prevention of osteoporosis through their effects on osteogenic precursors. In an example of sex steroid-specific effects, neural stem cells exhibit enhanced proliferation in response to estrogens, whereas androgens mediate inhibitory effects on their proliferation. Although stem cells can offer significant therapeutic benefits in various cardiovascular, neurodegenerative, endocrine disorders, and disorders of bone metabolism, a greater understanding of sex hormones on diverse stem cell populations is required to improve their ultimate clinical efficacy. In this review, we focus on the effects of estrogen and testosterone on various stem and progenitor cell types, and their relevant intracellular mechanisms.

Background Studies have noted gender differences in various models but have not investigated whether hormone depletion will abolish these differences. Therefore, we measured isometric force displacement in normal males, castrated males, normal females, and ovarectomized females. Materials and methods Adult male, adult female, castrated male, and ovarectomized female (250–350 g) Sprague Dawley rat pulmonary arteries (n = 7–8/group) were isolated and suspended in physiological organ baths. Force displacement was continuously recorded for 60 min of hypoxia. Data (mean ± SEM) was analyzed with two-way analysis of variance with post-hoc Bonferroni test or Student’s t-test. Results Maximum vasodilation of normal males was −79.47 ± 3.34%, while normal adult females exhibited a maximum vasodilation of −88.70 ± 6.21% (P = 0.8149). In addition, delayed, phase II vasoconstriction of male pulmonary arteries rings was 89.79 ± 7.25%, while adult females demonstrated a maximum phase II vasoconstriction of 95.90 ± 14.23% (P = 0.9342). Hormone depletion of males exhibited a maximum vasodilation of −70.45 ± 5.08% for castrated males as compared to −79.47 ± 3.34% for normal adult males (P = 0.3805). Castrated males exhibited a maximum phase II vasoconstriction of 86.20 ± 15.76% compared to 89.79 ± 7.25% exhibited by normal adult males (P = 0.9516). Conclusions Hormone depletion in males and females did not alter pulmonary vasoreactivity in acute hypoxia.

Stem cells may be a novel treatment modality for organ ischemia, possibly through beneficial paracrine mechanisms. Stem cells from older hosts have been shown to exhibit decreased function during stress. We therefore hypothesized that 1) neonatal bone marrow mesenchymal stem cells (nBMSCs) would produce different levels of IL-6, VEGF, and IGF-1 compared with adults (aBMSCs) when stimulated with TNF or LPS; 2) differences in cytokines would be due to distinct cellular characteristics, such as proliferation or pluripotent potential; and 3) differences in cytokines would be associated with differences in p38 MAPK and ERK signaling within nBMSCs. BMSCs were isolated from adult and neonatal mice. Cells were exposed to TNF or LPS with or without p38 or ERK inhibition. Growth factors were measured via ELISA, proliferation via daily cell counts, cell surface markers via flow cytometry, and pluripotent potential via alkaline phosphatase activity. nBMSCs produced lower levels of IL-6 and VEGF, but higher levels of IGF-1 under basal conditions, as well as after stimulation with TNF, but not LPS. Neonatal and adult BMSCs had similar pluripotent potentials and cell surface markers, but nBMSCs proliferated faster. Furthermore, p38 and ERK appeared to play a more substantial role in nBMSC cytokine and growth factor production. Neonatal stem cells may aid in decreasing the local inflammatory response during ischemia, and could possibly be expanded more rapidly than adult cells prior to therapeutic use.

Background Females demonstrate improved cardiac recovery after ischemia/reperfusion injury compared with males. Attenuation of myocardial dysfunction with preischemic estradiol suggests that estrogen may be an important mediator of this cardioprotection. However, it remains unclear whether post-injury estradiol may have clinical potential in the treatment of acute myocardial infarction. We hypothesize that postischemic administration of 17β-estradiol will decrease myocardial ischemia/reperfusion injury and improve left ventricular cardiac function. Materials and methods Adult male Sprague Dawley rat hearts ( n = 20) (Harlan, Indianapolis, IN) were isolated, perfused with Krebs-Henseleit solution via Langendorff model, and subjected to 15 min of equilibration, 25 min of warm ischemia, and 40 min reperfusion. Experimental hearts received postischemic 17β-estradiol infusion, 1 n m ( n = 4), 10 n m ( n = 4), 25 n m ( n = 4), or 50 n m ( n = 4), throughout reperfusion. Control hearts ( n = 4) were infused with perfusate vehicle. Results Postischemic recovery of left ventricular developed pressure was significantly greater with 1 n m (51.6% ± 7.4%) and 10 n m estradiol (47.7% ± 8.6%) than with vehicle (37.8% ± 9.7%) at end reperfusion. There was also greater recovery of the end diastolic pressure with 1 n m (47.8 ± 4.0 mmHg) and 10 n m estradiol (54.0 ± 4.0) compared with vehicle (75.3 ± 7.5). Further, 1 n m and 10 n m estrogen preserved coronary flow after ischemia and decreased coronary effluent lactated dehydrogenase compared with controls. Estrogen at 25 n m and 50 n m did not provide additional benefit in terms of functional recovery. Estrogen at all concentrations increased extracellular signal-regulated protein kinase phosphorylation. Conclusions Postischemic infusion of 17β-estradiol protects myocardial function and viability. The attractive potential for the clinical application of postischemic estrogen therapy warrants further study to elucidate the mechanistic pathways and differences between males and females.

Congestive heart failure is the most common cause of hospitalization in the United States for people over the age of sixty-five. As the population ages, the morbidity and mortality from heart failure will become more prevalent. Left heart failure has been, and continues to be, extensively studied. However, a recent report from the National Heart, Lung, and Blood Institute suggests that the right heart has been relatively under-investigated, and unfortunately, most of the basic mechanisms of intracellular signaling within the right heart still remain poorly understood. Right heart failure is now being increasingly recognized as distinctly different from left heart failure, and an important mediator of overall cardiovascular collapse. The purpose of this review, therefore, is to discuss the current understanding of right heart cellular development, physiology, and pathophysiology, as well as to review therapeutic interventions that are both currently available and under investigation.

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.

Signal transducer and activator of transcription (STAT) 3 is involved in mediating a broad range of biological processes, including cell survival, proliferation, and immune response. Recent evidence has indicated that STAT3 in cardiomyocytes can be activated by ischemic-oxidative stress and exerts cardioprotection in the ischemic heart. There is no information, however, regarding the effect of endothelial cell-derived STAT3 on the myocardial response to ischemiareperfusion (I/R) injury. We hypothesized that the ablation of the STAT3 gene in endothelial cells would worsen postischemic myocardial function by affecting capillary network integrity, suppressing antiapoptotic signaling. Isolated hearts from wild-type and endothelial cell STAT3 knockout (STAT3KO) mice were subjected to 20 min of global ischemia followed by 60 min of reperfusion. Endothelial cell STAT3 deficiency decreased recovery of myocardial function in response to I/R, which was associated with higher levels of LDH release, decreased activation of myocardial STAT3, and elevated p38 MAPK activation in STAT3 endothelial knockout (KO) hearts. In addition, although no significant apoptosis was observed in wild-type and KO hearts, our results showed more expression of myocardial caspase-8 and more apoptosis in the myocardium around the capillary in STAT3KO mice subjected to I/R. Furthermore, endothelial cell STAT3 ablation resulted in increased myocardial expression of IL-6 and suppressor of cytokine signal 3. This study demonstrates that endothelial cell-derived STAT3 plays an important role in postischemic myocardial function.

Necrotizing enterocolitis (NEC) is an emergency of the newborn that often requires surgery. Growth factors from stem cells may aid in decreasing intestinal damage while also promoting restitution. We hypothesized that 1) TNF, LPS, or hypoxia would alter bone marrow mesenchymal stem cell (BMSC) TNF, IGF-1, IL-6, and VEGF production, and 2) TNF receptor type 1 (TNFR1) or type 2 (TNFR2) ablation would result in changes to the patterns of cytokines and growth factors produced. BMSCs were harvested from female wild-type (WT), TNFR1 knockout (KO), and TNFR2KO mice. Cells were stimulated with TNF, LPS, or hypoxia. After 24 h, cell supernatants were assayed via ELISA. Production of TNF and IGF-1 was decreased in both knockouts compared with WT regardless of the stimulus utilized, whereas IL-6 and VEGF levels appeared to be cooperatively regulated by both the activated TNF receptor and the initial stimulus. IL-6 was increased compared with WT in both knockouts following TNF stimulation but was significantly decreased with LPS. Compared with WT, hypoxia increased IL-6 in TNFR1KO but not TNFR2KO cells. TNF stimulation decreased VEGF in TNFR2KO cells, whereas TNFR1 ablation resulted in no change in VEGF compared with WT. TNFR1 ablation resulted in a decrease in VEGF following LPS stimulation compared with WT; no change was noted in TNFR2KO cells. With hypoxia, TNFR1KO cells expressed more VEGF compared with WT, whereas no difference was noted between WT and TNFR2KO cells. TNF receptor ablation modifies BMSC cytokine production. Identifying the proper stimulus and signaling cascades for the production of desired growth factors may be beneficial in maximizing the therapeutic potential of stem cells.

Sex differences exist in a variety of cardiovascular disorders. Sex hormones have been shown to mediate pulmonary artery (PA) vasodilation. However, the effects of fluctuations in physiological sex hormone levels due to sex and menstrual cycle on PA vasoreactivity have not been clearly established yet. We hypothesized that sex and menstrual cycle affect PA vasoconstriction under both normoxic and hypoxic conditions. Isometric force displacement was measured in isolated PA rings from proestrus females (PF), estrus and diestrus females (E/DF), and male (M) Sprague-Dawley rats. The vasoconstrictor response under normoxic conditions (organ bath bubbled with 95% O2-5% CO2) was measured after stimulation with 80 mmol/l KCl and 1 μmol/l phenylephrine. Hypoxia was generated by changing the gas to 95% N2-5% CO2. PA rings from PF demonstrated an attenuated vasoconstrictor response to KCl compared with rings from E/DF (75.58 ± 3.2% vs. 92.43 ± 4.24%, P < 0.01). Rings from M also exhibited attenuated KCl-induced vasoconstriction compared with E/DF (79.34 ± 3.2% vs. 92.43 ± 4.24%, P < 0.05). PA rings from PF exhibited an attenuated vasoconstrictor response to phenylephrine compared with E/DF (59.61 ± 2.98% vs. 70.03 ± 4.61%, P < 0.05). While the maximum PA vasodilation during hypoxia did not differ between PF, E/DF, and M, phase II of hypoxic pulmonary vasoconstriction was markedly diminished in the PA from PF (64.10 ± 7.10% vs. 83.91 ± 5.97% in M, P < 0.05). We conclude that sex and menstrual cycle affect PA vasoconstriction in isolated PA rings. Even physiological increases in circulating estrogen levels attenuate PA vasoconstriction under both normoxic and hypoxic conditions.

Background Gender differences exist in the myocardial response to acute ischemia/reperfusion (I/R) injury and may be attributed to the effects of the sex hormones estrogen and testosterone. The role of estrogen in myocardial injury has been extensively studied but little information exists regarding the myocardial involvement of testosterone. Based on the deleterious effects of chronic endogenous and acute testosterone exposure observed in our previous studies, we postulated that chronic exogenous testosterone administration would also exhibit deleterious effects on myocardial function following I/R. Methods Langendorff perfused rat hearts were subjected to 25 min ischemia, 40 min reperfusion, and left ventricular developed pressure (LVDP) was recorded. Control and 5α-dihydrotestosterone (DHT) treated groups each consisted of normal males, castrated males, ovariectomized (OVX) females, and senescent females. P Results Chronic DHT replacement therapy showed no difference in functional ischemic recovery as measured by LVDP after 40 min reperfusion in castrated males (65.1 ± 8.13% versus 66.3 ± 4.54%), OVX females (64.5 ± 10.6% versus 50.2 ± 5.97%), and senescent females (42.1 ± 0.04% versus 41 ± 0.05%). Interestingly, LVDP was greater in DHT treated males than control males after I/R (65.2 ± 8.20% versus 47.6 ± 5.19%). Also, DHT treatment resulted in significantly increased recovery of LVDP after only 10 min reperfusion in castrated males, OVX females, and senescent females compared with their untreated counterparts (54.8 ± 11.9% versus 32.9 ± 5.75%, 66.7 ± 11.5% versus 43.1 ± 8.15%, 53.4 ± 10.1% versus 32.9 ± 5.75%, respectively). Conclusion Contrary to the adverse effects we observed in earlier studies with both endogenous and brief exogenous testosterone in myocardium injured by I/R, the present study revealed that chronic exogenous testosterone neither improved nor worsened myocardial functional recovery following 25 min ischemia and 40 min reperfusion.

The mechanisms by which mesenchymal stem cells (MSCs) may protect native tissue are incompletely understood. Understanding the mechanisms by which these cells release factors such as vascular endothelial growth factor (VEGF), may lead to enhanced protection. We hypothesized that stress, in the form of hypoxia or TNF, activates MSCs to release VEGF by STAT3 and p38 MAPK dependent mechanisms. Mouse MSCs from wild type (WT) and STAT3 knockout mice (STAT3KO) were harvested and purified by a single-step method using adhesion. The release of VEGF was analyzed by using MSC conditioned media under hypoxia or TNF stimulation with or without p38 MAPK inhibition. Activation of STAT3 and p38 MAPK was determined by analysis of cell lysates. MSCs released VEGF under normoxia, which was associated with constitutive STAT3 activity. STAT3 deficiency resulted in decreased MSC production of VEGF. In response to hypoxia or TNF, MSCs produced more VEGF, which was correlated with hypoxia or TNF activated p38 MAPK and STAT3. The p38 MAPK inhibitor significantly decreased hypoxia-induced or TNF-stimulated VEGF production in WT. Additionally, STAT3 ablation neutralized hypoxia-induced MSC release of VEGF. No effect of p38 MAPK inhibitor alone was observed on MSC release of VEGF in WT. However, inhibition of p38 MAPK blocked release of VEGF in STAT3KO MSCs. MSCs are a potent source of VEGF, the production of which is mediated by STAT3 under normoxia partly; however, following hypoxia or TNF exposure, MSC release of VEGF is mediated by both STAT3 and p38 MAPK.

Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline in renal function. Although several inflammatory mediators have been implicated in the pathophysiology of renal obstruction, the contribution of TNF-α to obstruction-induced fibrosis and renal dysfunction has not been thoroughly evaluated. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1) every 84 h. The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were analyzed for TNF-α expression (ELISA), macrophage infiltration (ED-1 staining), transforming growth factor-β1expression (ELISA, RT-PCR), collagen I and IV activity (Western Blot, immunohistochemistry), α-smooth muscle actin accumulation (immunohistochemistry, Western blot analysis), and angiotensinogen expression (Western blot). In a separate arm, the glomerular filtration rate (inulin clearance) of rats subjected to unilateral ureteral obstruction in the presence of either vehicle or PEG-sTNFR1 was determined. Renal obstruction induced increased tissue TNF-α and transforming growth factor-β1levels, collagen I and IV activity, interstitial volume, α-smooth muscle actin accumulation, angiotensinogen expression, and renal dysfunction, whereas treatment with PEG-sTNFR1 significantly reduced each of these markers of renal fibrosis. These results demonstrate that TNF-α mediates obstruction-induced renal fibrosis and identify TNF-α neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.

Bacteria have developed mechanisms to sequester host iron via chelators such as deferoxamine (DFO). Interestingly, DFO has been shown to stimulate acute intestinal epithelial cell inflammatory cytokine production in the absence of bacteria; however, this mechanism has not been elucidated. Intestinal epithelial cell production of IL-6 and TNF-α is elevated in various gastrointestinal pathologies, including acute intestinal ischemia. Similarly, VEGF and HGF are essential to intestinal epithelial cell integrity. Therapeutic strategies that decrease IL-6 and TNF-α while increasing VEGF and HGF therefore have theoretical appeal. We hypothesized that 1) fetal human intestinal epithelial cells acutely produce increased IL-6, TNF-α, VEGF, and HGF during iron chelation and 2) the MAPK pathway mediates these effects. Fetal human intestinal epithelial cells were stimulated by iron chelation (1 mM DFO) with and without p38 MAPK, ERK, or JNK inhibition. Supernatants were harvested after 24 h of incubation, and IL-6, TNF-α, VEGF, and HGF levels were quantified by ELISA. Activation of MAPK pathways was confirmed by Western blot analysis. DFO stimulation resulted in a significant increase in epithelial cell IL-6 and VEGF production while yielding a decrease in HGF production ( P < 0.05). Unexpectedly, TNF-α was not detectable. p38 MAPK, ERK, and JNK inhibition significantly decreased IL-6, VEGF, and HGF production ( P < 0.05). In conclusion, DFO acutely increases fetal human intestinal epithelial cell IL-6 and VEGF expression while causing an unexpected decrease in HGF expression and no detectable TNF-α production. Furthermore, chelator-induced intestinal epithelial cell cytokine expression depends on p38, ERK, and JNK MAPK pathways.