Nathan M. Novotny

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.

Sex steroids and stem cell function.

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.

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.

TGF-α increases human mesenchymal stem cell-secreted VEGF by MEK- and PI3-K- but not JNK- or ERK-dependent mechanisms

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.

β-Blockers in sepsis: Reexamining the evidence

Sepsis remains the leading cause for noncardiac intensive care unit deaths in the United States. Despite recent advances in the treatment of this devastating condition, mortality and morbidity remain unacceptably high. Sepsis is characterized by a multitude of pathophysiological changes that include inflammation, metabolic derangements, hemodynamic alterations, and multiorgan dysfunction. Unfortunately, several studies of treatment modalities aimed at correcting one or more of the underlying derangements have led to disappointing results. New treatment modalities are needed. &bgr;-Receptor blockers have long been used for a variety of conditions such as coronary artery disease, congestive heart failure, and arterial hypertension. Recent data suggest that &bgr;-blocker effects on metabolism, glucose homeostasis, cytokine expression, and myocardial function may be beneficial in the setting of sepsis. Although treating a potentially hypotensive condition with a drug with antihypertensive properties may initially seem counterintuitive, the metabolic and immunomodulatory properties of &bgr;-blockers may be of benefit. It is the purpose of this review to discuss the effects of &bgr;-blockers on the following: (1) metabolism, (2) glucose regulation, (3) the inflammatory response, (4) cardiac function, and (5) mortality in sepsis.

Stem cells as a potential future treatment of pediatric intestinal disorders.

All surgical disciplines encounter planned and unplanned ischemic events that may ultimately lead to cellular dysfunction and death. Stem cell therapy has shown promise for the treatment of a variety of ischemic and inflammatory disorders where tissue damage has occurred. As stem cells have proven beneficial in many disease processes, important opportunities in the future treatment of gastrointestinal disorders may exist. Therefore, this article will serve to review the different types of stem cells that may be applicable to the treatment of gastrointestinal disorders, review the mechanisms suggesting that stem cells may work for these conditions, discuss current practices for harvesting and purifying stem cells, and provide a concise summary of a few of the pediatric intestinal disorders that could be treated with cellular therapy.

Preoperative prediction of need for fundoplication before gastrostomy tube placement in children.

PURPOSE Percutaneous endoscopic gastrostomy (PEG) tube placement is a common procedure performed for children with oral aspiration and failure to thrive. The concurrent presence of gastroesophageal reflux (GER) may be difficult to diagnose in these children and may dictate the need for an antireflux procedure. The purpose of this study was to review our preoperative evaluation of children undergoing PEG placement to better elucidate preoperative factors that may require eventual fundoplication. METHODS A retrospective review at a tertiary care, childrens hospital between May 2002 and August 2007 was performed of patients undergoing PEG placement. Patients were identified through database search by operative procedure codes. Patient groups were defined as those undergoing PEG alone (group 1) and those requiring fundoplication after prior PEG (group 2). Comparison of patient demographics and radiologic qualitative results of GER was analyzed using chi(2) analysis, with significance determined at P < .05. RESULTS A total of 863 patients underwent PEG placement over this 64-month period. A sampled cohort of patients undergoing PEG over a year comprised group 1. Forty-four patients (5.1%) underwent Nissen fundoplication after prior PEG placement (group 2). Patient demographics were similar between the groups. Comparison of comorbid conditions and qualitative indicators of GER between the groups showed only cerebral palsy had a significantly higher associated risk of GER that required antireflux surgery. Preoperative clinical assessment had a 95% positive predictive value in identifying children who required only PEG. CONCLUSIONS Despite the high predictive value of individualized clinical assessment in the ultimate decision for gastrostomy without need of fundoplication, further studies are needed to determine whether children with conditions such as cerebral palsy may require a concurrent antireflux surgery at the time of gastrostomy.

Percutaneous endoscopic gastrostomy buttons in children: superior to tubes☆

BACKGROUND There is a paucity of literature comparing outcomes of percutaneous endoscopic gastrostomy (PEG) tubes vs PEG buttons. Primary PEG buttons offer an advantage of being a single-step low-profile enteral access device with potentially fewer complications. METHODS A retrospective review of patients undergoing PEG tubes and buttons (January 2006-August 2007) was performed. Power analysis demonstrated that 105 patients in each group were needed. Patient characteristics were collected in each group and evaluated by chi(2) and t tests. P values of less than .05 were considered significant. RESULTS A total of 223 children having undergone PEG (110 tubes, 113 buttons) were identified. No differences were found in operative time, intraoperative complications, clogging, breakage, infections, emergency department visits, or hospital readmissions. However, children undergoing PEG button placement were more likely to spend only one night in the hospital vs PEG tube (60% vs 25%, respectively; P < .001). In addition, PEG buttons had fewer dislodgements (4 vs 15; P < .05). CONCLUSION The PEG buttons are less likely to become dislodged than PEG tubes. Infection rates were not found to be different between groups. Children with PEG buttons were more likely to be discharged earlier than children with PEG tubes. Primary PEG buttons are clinically comparable to PEG tubes with less concern for dislodgements.

Angiopoietin-1 in the treatment of ischemia and sepsis.

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.

MEK mediates the novel cross talk between TNFR2 and TGF-EGFR in enhancing vascular endothelial growth factor (VEGF) secretion from human mesenchymal stem cells

BACKGROUND Bone marrow mesenchymal stem cells (MSCs) may mediate their beneficial effects by paracrine mechanisms. Recently, we reported that tumor necrosis factor-alpha (TNF-alpha) increased the release of vascular endothelial growth factor (VEGF) from human MSCs and augmented transforming growth factor-alpha (TGF-alpha)-stimulated VEGF secretion. However, it is unknown whether TNF-alpha stimulates VEGF production via TNF receptor 1 (TNFR1) or 2 (TNFR2) and the mechanism by which TNF-alpha augments TGF-alpha (a ligand of epidermal growth factor receptor, EGFR) stimulated VEGF production. We hypothesized that the ablation of TNFR2 would decrease TNF-alpha-stimulated and/or TGF-alpha- stimulated VEGF production via MEK-dependent mechanisms. METHODS MSCs transfected with TNFR1, TNFR2, or GAPDH siRNA were stimulated with TNF-alpha and/or TGF-alpha for 24 h. VEGF levels in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). A Western blot analysis was performed to measure the activation of MEK and ERK and the expression of TNFR1 and TNFR2. RESULTS TNF-alpha or TGF-alpha increased VEGF secretion in cells transfected with GAPDH or TNFR1 siRNA. The combination of TNF-alpha and TGF-alpha increased VEGF production. TNF-alpha and/or TGF-alpha stimulation increased phospho-MEK and phospho-ERK in cells transfected with TNFR1 siRNA. Conversely, the effects of TNF-alpha and/or TGF-alpha on MSC VEGF production were significantly decreased, and MEK/ERK activation was negated in cells transfected TNFR2 siRNA. CONCLUSION TNFR2 plays a vital role in the effects of TNF-alpha and TGF-alpha on MSC VEGF production. The activation of MEK was implicated in this novel cross talk between TNFR2 and TGF-alpha-EGFR in regulating the production of VEGF in human MSCs.