Paul R. Crisostomo

Human mesenchymal stem cells stimulated by TNF-α, LPS, or hypoxia produce growth factors by an NFκB- but not JNK-dependent mechanism

Understanding the mechanisms by which adult stem cells produce growth factors may represent an important way to optimize their beneficial paracrine and autocrine effects. Components of the wound milieu may stimulate growth factor production to promote stem cell-mediated repair. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), endotoxin (LPS), or hypoxia may activate human mesenchymal stem cells (MSCs) to increase release of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1), or hepatocyte growth factor (HGF) and that nuclear factor-kappa B (NF kappa B), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) mediates growth factor production from human MSCs. To study this, human MSCs were harvested, passaged, divided into four groups (100,000 cells, triplicates) and treated as follows: 1) with vehicle; 2) with stimulant alone [24 h LPS (200 ng/ml), 24 h TNF-alpha (50 ng/ml), or 24 h hypoxia (1% O2)]; 3) with inhibitor alone [NF kappa B (PDTC, 1 mM), JNK (TI-JIP, 10 microM), or ERK (ERK Inhibitor II, 25 microM)]; and 4) with stimulant and the various inhibitors. After 24 h incubation, MSC activation was determined by measuring supernatants for VEGF, FGF2, IGF-1, or HGF (ELISA). TNF-alpha, LPS, and hypoxia significantly increased human MSC VEGF, FGF2, HGF, and IGF-1 production versus controls. Stem cells exposed to injury demonstrated increased activation of NF kappa B, ERK, and JNK. VEGF, FGF2, and HGF expression was significantly reduced by NF kappa B inhibition (50% decrease) but not ERK or JNK inhibition. Moreover, ERK, JNK, and NF kappa B inhibitor alone did not activate MSC VEGF expression over controls. Various stressors activate human MSCs to increase VEGF, FGF2, HGF, and IGF-1 expression, which depends on an NFkB mechanism.

High passage number of stem cells adversely affects stem cell activation and myocardial protection.

ABSTRACT Progenitor cell plasticity enhances positive remodeling of damaged tissue. We and others have previously shown that progenitor cells may limit apoptosis and modulate inflammation in part by the production of growth factors. However, recent studies suggest that progenitor cells senesce and lose their differentiation potential with increasing time in culture and passage. We hypothesize that murine bone marrow mesenchymal stem cells (MSCs) are cardioprotective against ischemia/reperfusion injury in the isolated perfused rat heart, and that passage number has an adverse effect on MSC activation and cardioprotection. Adult male and female Sprague-Dawley rat hearts were isolated, perfused via Langendorff model, and subjected to ischemia/reperfusion. Mouse MSCs were harvested, cultured, suspended in perfusate, and infused before global index ischemia. Hearts were assigned to controls or infusion with passage 3, 5, or 10 MSCs. In addition, MSCs in culture were stressed by hypoxia and increasing doses of endotoxin (lipopolysaccharide). Mesenchymal stem cell activation was determined by measuring vascular endothelial growth factor production with enzyme-linked immunosorbent assay. All data are reported as mean ± SEM and were analyzed with 2-way analysis of variance. Differences are considered significant if P < 0.05. Passage 3 murine MSC infusion in hearts before ischemia reduced the depression of left ventricular developed pressure, attenuated the increase of end-diastolic pressure, and reduced the depression of +dP/dT and −dP/dT. However, the MSC protective effect disappeared in hearts infused with passage 5 and passage 10 MSCs. Although hypoxia and lipopolysaccharide resulted in significant activation of MSCs, passage 3 MSCs demonstrated significantly greater vascular endothelial growth factor release than passage 5 and 10 MSCs. Acute murine MSC infusion confers protection in isolated rat hearts. However, high passage number has an adverse effect on MSC activation and protection. This portends limited ex vivo expansion before possible therapeutic use.

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.

Cytokines in necrotizing enterocolitis.

ABSTRACT Necrotizing enterocolitis (NEC) is a devastating intra-abdominal emergency in the newborn period. The disease involves bowel wall inflammation, ischemic necrosis, eventual perforation, and the need for urgent surgical intervention. Unrecognized or left untreated, the neonate can decompensate quickly, often progressing to shock, multisystem organ failure, and eventual death. During the past several years, a number of basic science and clinical trials have been established in an attempt to understand the pathophysiology of NEC. As many researchers feel that NEC develops as an uncontrolled inflammatory response that leads to intestinal ischemia, a large number of studies have been focused on the inflammatory cascade and the role that cytokines play within that cascade. Although a large amount of data has been generated from these studies, the events leading to the ischemic injury of the intestine are still not fully understood. This article will therefore focus on the key cytokines involved with NEC, in an attempt to present the current literature and studies that support their involvement.

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.

Pretreatment with adult progenitor cells improves recovery and decreases native myocardial proinflammatory signaling after ischemia

ABSTRACT Cardiogenic shock from myocardial ischemia is the leading cause of death of both men and women. Although adult progenitor cells have emerged as a potential therapy for heart disease, reports indicate that transplanted adult progenitor cells may not differentiate into heart muscle. We hypothesized that pretreatment with adult progenitor cells may protect myocardium from acute ischemic damage. Treatment immediately before an ischemic event removes the possibility that differentiation to heart muscle may account for the observed effects. In the present study, we determined that adult progenitor cells from three different sources (human bone marrow, rat bone marrow, and human adipose tissue) immediately protect native myocardium against ischemia and decrease myocardial proinflammatory and proapoptotic signaling. Postischemic recovery of adult progenitor cell-pretreated hearts was significantly better than that of control hearts. This was correlated with a 50% decrease in proinflammatory cytokine production. The use of a differentiated cell control had no such effect. Therefore, adult progenitor cell pretreatment improved postischemic myocardial function, decreased myocardial production of inflammatory mediators, and limited proapoptotic signaling. These results represent the first demonstration that pretreatment with progenitor cells is myocardial protective. These findings may not only have mechanistic implications regarding the benefit of progenitor cells but may also have clinical therapeutic implications before planned ischemic events.

Embryonic stem cells attenuate myocardial dysfunction and inflammation after surgical global ischemia via paracrine actions

Stem cell treatment may positively influence recovery and inflammation after shock by multiple mechanisms, including the paracrine release of protective growth factors. Embryonic stem cells (ESCs) are understudied and may have greater protective power than adult bone marrow stem cells (BMSCs). We hypothesized that ESC paracrine protective mechanisms in the heart (decreased injury by enhanced growth factor-mediated reduction of proinflammatory cytokines) would be superior to the paracrine protective mechanisms of the adult stem cell population in a model of surgically induced global ischemia. Adult Sprague-Dawley rat hearts were isolated and perfused via Langendorff model. Hearts were subjected to 25 min of warm global ischemia and 40 min of reperfusion and were randomly assigned into one of four groups: 1) vehicle treated; 2) BMSC or ESC preischemic treatment; 3) BMSC or ESC postischemic treatment; and 4) BMSC- or ESC-conditioned media treatment. Myocardial function was recorded, and hearts were analyzed for expression of tissue cytokines and growth factors (ELISA). Additionally, ESCs and BMSCs in culture were assessed for growth factor production (ELISA). ESC-treated hearts demonstrated significantly greater postischemic recovery of function (left ventricular developed pressure, end-diastolic pressure, and maximal positive and negative values of the first derivative of pressure) than BMSC-treated hearts or controls at end reperfusion. ESC-conditioned media (without cells) also conferred cardioprotection at end reperfusion. ESC-infused hearts demonstrated increased VEGF and IL-10 production compared with BMSC hearts. ESC hearts also exhibited decreased proinflammatory cytokine expression compared with MSC hearts. Moreover, ESCs in cell culture demonstrated greater pluripotency than MSCs. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells.

Surgically relevant aspects of stem cell paracrine effects

Stem cells are undifferentiated, self-renewing, and multipotent (able to differentiate into multiple cell types). Unlike traditional treatment modalities, these unique characteristics may enable stem cells to undo irreversible cellular damage and rebuild injured or diseased tissue. Recent evidence suggests that stem cells may influence positively the recovery from injury via paracrine factors that promote tissue repair. Insights into paracrine mechanisms of stem cells may help the surgeon scientist devise more effective therapies and earlier widespread application. This review: (1) unravels the pathways for stem cell mediated paracrine protection; (2) highlights the growth factors and cytokines expressed; (3) and explores the potential of using stem cells clinically.

Jak/STAT/SOCS signaling circuits and associated cytokine-mediated inflammation and hypertrophy in the heart.

ABSTRACT Cytokines are important mediators of cardiac disease. Accumulating evidence indicates that members of the interleukin-6 family of cytokines promote cardiac hypertrophy through the activation of the Janus kinase-signal transducer and activator of transcription (Jak/STAT) pathway. Aberrant Jak/STAT signaling may promote progression from hypertrophy to heart failure. Suppressor of cytokine signaling (SOCS) proteins are underexplored, negative regulators of Jak/STAT signaling. SOCS proteins may also interact with other inflammatory pathways known to affect cardiac function. A better understanding of the therapeutic potential of these proteins may lead to the controlled progression of heart failure and the limitation of myocardial depression. This review summarizes the cardiophysiological effect of the IL-6 cytokine family, outlines the mechanistic pathway of Jak/STAT signaling, explores the regulatory role of SOCS proteins in the heart, and discusses the potential of using SOCS proteins clinically.

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.