Ian B. Copland

Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing

Human mesenchymal stromal cells (MSC) can suppress T-cell activation in vitro in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. However, their clinical effects on immune ailments have been inconsistent, with a recent phase III study showing no benefit in acute graft-versus-host disease (GvHD). We here tested the hypothesis that the banked, cryopreserved MSC often used in clinical trials display biologic properties distinct from that of MSC in the log phase of growth typically examined in pre-clinical studies. In freshly thawed cryopreserved MSC derived from normal human volunteers, we observed that MSC up-regulate heat-shock proteins, are refractory to interferon (IFN)-γ-induced up-regulation of IDO, and are compromised in suppressing CD3/CD28-driven T cell proliferation. Immune suppressor activity, IFN-γ responsiveness and induction of IDO were fully restored following 24 h of MSC tissue culture post-thaw. These results highlight a possible cause for the inefficacy of MSC-based immunotherapy reported in clinical trials using cryopreserved MSC thawed immediately prior to infusion.

Stretch-activated signaling pathways responsible for early response gene expression in fetal lung epithelial cells.

High‐tidal volume ventilation has been shown to increase the expression of several inflammation‐associated genes prior to overt physiologic lung injury. Herein, using an in vitro stretch system, we investigated the mechanotransduction pathways involved in ventilation‐induced expression of these early response genes (i.e., early growth response gene (Egr)1, heat‐shock protein (HSP)70, and the pro‐inflammatory cytokines interleukin (IL)‐1β, IL‐6, and MIP‐2). Mechanical stretch of fetal lung epithelial cells activated various signaling pathways, resulting in transient or progressive increases in gene expression of the early response genes. The transient increase in Egr1 and IL‐6 expression was mediated via p44/42 mitogen‐activated protein kinase (p44/42 MAPK), while nuclear factor‐kappaB (NF‐κB) was responsible for the sustained and progressive increase in expression of HSP70 and MIP‐2. Blockage of Egr‐1 expression did not affect the upregulation of IL‐6, HSP70, MIP‐2, and itself by stretch. Inhibition of calcium mobilization abolished stretch‐induced p44/42 MAPK activation and NF‐κB nuclear translocation as well as increased expression of all early response genes. Similar results were obtained with an inhibitor of Ras. These results suggest that mechanical stretch of fetal lung epithelial cells evokes a complex network of signaling molecules, which diverge downstream to regulate the temporal expression of a unique set of early response genes, but upstream converge at calcium. Thus, calcium mobilization may be a point of hierarchical integration of mechanotransduction in lung epithelial cells. J. Cell. Physiol. 210: 133–143, 2007.

Down-regulation of sonic Hedgehog expression in pulmonary hypoplasia is associated with congenital diaphragmatic hernia

The pathogenesis of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) is unknown. The sonic hedgehog (Shh) cascade is crucial for the patterning of the early respiratory system in mice. To establish whether Shh plays a role in the pathogenesis of lung hypoplasia in CDH, we investigated the gestation-specific expression of Shh in normal rat and human lungs using in situ hybridization and immunohistochemistry. The expression pattern was compared with that of age-matched samples of hypoplastic lungs associated with CDH in humans and in the 2,4-dichlorophenyl-p-nitrophenylether (nitrofen) rat model. Our results showed that in normal controls the expression of Shh increased with advancing gestation, peaked in the late pseudoglandular stage, and declined thereafter. The expression of Shh is initially down-regulated in pulmonary hypoplasia associated with CDH and peaks instead during the late canalicular stage. These data indicate that maximal expression of Shh occurs when respiratory bronchioles develop and thinning of the interstitium takes place, suggesting that Shh may play a role in these processes. Furthermore, we observed that Shh inhibited fetal lung fibroblast proliferation in vitro. Therefore, it is tempting to speculate that alterations in Shh expression may affect these developmental processes, thereby contributing to the pulmonary abnormality in CDH.

Evidence for Transcriptional Regulation of the Glucose‐6‐Phosphate Transporter by HIF‐1α: Targeting G6PT with Mumbaistatin Analogs in Hypoxic Mesenchymal Stromal Cells

Mesenchymal stromal cell (MSC) markers are expressed on brain tumor‐initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose‐6‐phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O2) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose‐6‐phosphatase catalytic subunit β, whereas expression of the glucose‐6‐phosphatase catalytic subunit α and the islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)‐1α and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF‐1α. Gene silencing of HIF‐1α prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF‐1α resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC‐HIF‐1α cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors. STEM CELLS 2009;27:489–497

Mesenchymal stromal cells genetically engineered to overexpress IGF-I enhance cell-based gene therapy of renal failure-induced anemia

We previously demonstrated that erythropoietin (EPO)-secreting mesenchymal stromal cells (MSC) can be used for the long-term correction of renal failure-induced anemia. The present study provides evidence that coimplantation of insulin-like growth factor I (IGF-I)-overexpressing MSC (MSC-IGF) improves MSC-based gene therapy of anemia by providing paracrine support to EPO-secreting MSC (MSC-EPO) within a subcutaneous implant. IGF-I receptor RNA expression in murine MSC was demonstrated by RT-PCR. Functional protein expression was confirmed by immunoblots and MSC responsiveness to IGF-I stimulation in vitro. IGF-I was also shown to improve MSC survival following staurosporin-induced apoptosis in vitro. A cohort of C57Bl/6 mice was rendered anemic by right kidney electrocoagulation and left nephrectomy. MSC-EPO were subsequently admixed in a bovine collagen matrix and implanted, in combination with MSC-IGF or MSC null, by subcutaneous injection in renal failure mice. In mice receiving MSC-EPO coimplanted with MSC-IGF, hematocrit elevation was greater and enhanced compared with control mice; heart function was also improved. MSC-IGF coimplantation, therefore, represents a promising new strategy for enhancing MSC survival within implanted matrices and for improving cell-based gene therapy of renal anemia.

Cyclooxygenase inhibition in ventilator-induced lung injury.

BACKGROUND:We tested the hypothesis that inhibition of cyclooxygenase (COX) attenuates in vivo ventilator-induced lung injury (VILI) in a prospective, randomized laboratory investigation in a university-affiliated laboratory. Adult male rats were anesthetized and randomized with or without nonselective COX inhibition (ibuprofen) and were subjected to injurious mechanical ventilation (positive end-expiratory pressure = 0; peak inspiratory pressure = 21 mm Hg). METHODS:We investigated the profile of VILI (respiratory mechanics, cytokines, eicosanoids), expression of COX enzymes, and activation of nuclear factor (NF)-&kgr;B in ibuprofen- versus vehicle-treated animals. Injurious ventilation caused lung injury (i.e., decrement in compliance, tissue edema, and elevated inflammatory cytokines, eicosanoids, and COX-2). RESULTS:Pretreatment with ibuprofen that effectively inhibited eicosanoid synthesis and COX-2 activity increased survival and attenuated lung edema and decrement in respiratory mechanics. Ibuprofen had no modulatory effect on ventilator-induced activation of NF-&kgr;B or inflammatory cytokines (tumor necrosis factor-&agr;, interleukin [IL]-1&bgr;, IL-6, GRO/KC [growth-related oncogene/keratinocyte chemoattractant]). COX activity seems important in the pathogenesis of VILI in the in vivo rat. Inhibition of COX provides significant protection (i.e., survival, pulmonary function) in VILI, but without affecting levels of important mediators (tumor necrosis factor-&agr;, IL-1&bgr;, IL-6, GRO/KC) or activation of NF-&kgr;B. CONCLUSIONS:These data confirm that nonselective COX inhibition provides partial protection against VILI and that the NF-&kgr;B signaling pathway is not exclusively eicosanoid dependent. Studies of COX inhibition in ventilator-associated lung injury might benefit from multimodal targeting that includes a comprehensive focus on inflammatory cytokines and NF-&kgr;B.