Jessica Cuerquis

Improved Autograft Survival of Mesenchymal Stromal Cells by Plasminogen Activator Inhibitor 1 Inhibition

Mesenchymal stromal cells (MSCs) display robust reparative properties through their ability to limit apoptosis, enhance angiogenesis, and direct positive tissue remodeling. However, low in vivo survival of transplanted cells limits their overall effectiveness and significantly affects their clinical usage. Consequently, identifying strategies to improve cell survival in vivo are a priority. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue, such as infarcted myocardium, where there is poor blood supply and low oxygen tension. Therefore, we examined how MSCs respond to a hypoxic, nutrient‐poor stress environment to identify trophic factors that could be manipulated in advance of MSC transplantation. Combining microarray and proteomic screens we identified plasminogen activator inhibitor 1 (PAI‐1) as one factor consistently upregulated in our in vitro ischemia‐mimicking conditions. Subsequent genetic and chemical manipulation studies define PAI‐1 as a negative regulator of MSC survival in vivo. Mechanistically, MSC‐derived PAI‐1 does not alter MSC survival through a plasmin‐dependent mechanism but rather directly impacts on the adhesiveness of MSCs to their surrounding matrices. Thus we can conclude that post‐transplantation, PAI‐1 negatively impacts MSC survival by promoting anoikis via matrix detachment. STEM CELLS 2008;27:467–477

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation.

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α-pretreated human bone marrow-derived MSCs on resting or activated T cells. METHODS MSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed. RESULTS Unprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions. CONCLUSIONS Unprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.

Coupling erythropoietin secretion to mesenchymal stromal cells enhances their regenerative properties

AIMS Mesenchymal stromal cells (MSCs) possess intrinsic features that identify them as useful for treating ischaemic syndromes. Poor in vivo survival/engraftment of MSCs, however, limits their overall effectiveness. In this work, we tested whether genetically engineering MSCs to secrete erythropoietin (Epo) could represent a better therapeutic platform than MSCs in their native form. METHODS AND RESULTS MSCs from C57Bl/6 mice were retrovirally transduced with either an empty vector or one that causes the production of Epo and were then analysed for the alterations in angiogenic and survival potential. Using a mouse model of myocardial infarction (MI), the regenerative potential of null MSCs and Epo-overexpressing MSCs (Epo+MSCs) was assessed using serial echocardiogram and invasive haemodynamic measurements. Infarct size, capillary density and neutrophil influx were assessed using histologic techniques. Using in vitro assays coupled with an in vivo Matrigel plug assay, we demonstrate that engineering MSCs to express Epo does not alter their immunophenotype or plasticity. However, relative to mock-modified MSCs [wild-type (WT)-MSCs], Epo+MSCs are more resilient to apoptotic stimuli and initiate a more robust host-derived angiogenic response. We also identify and characterize the autocrine loop established on MSCs by having them secrete Epo. Furthermore, in a murine model of MI, animals receiving intracardiac injections of Epo+MSCs exhibited significantly enhanced cardiac function compared with WT-MSCs and saline-injected control animals post-MI, owing to the increased myocardial capillary density and the reduced neutrophilia. CONCLUSION Epo overexpression enhances the cellular regenerative properties of MSCs by both autocrine and paracrine pathways.

Monocyte Derivatives Promote Angiogenesis and Myocyte Survival in a Model of Myocardial Infarction

In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-α, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling.

Hierarchical scaffold design for mesenchymal stem cell-based gene therapy of hemophilia B.

Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite-PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatment of hemophilia B and other plasma protein deficiencies.

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.

A Fusion of GMCSF and IL-21 Initiates Hypersignaling Through the IL-21Rα Chain With Immune Activating and Tumoricidal Effects In Vivo

We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Rα-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21s aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-α, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Rα+ lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Rα+ lymphocytes, we tested its cytolytic effect on IL-21Rα+ EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Rα agonist that co-opts IL-21Rα-dependent signaling in a manner permissive for targeted cancer immunotherapy.We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Ralpha-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21s aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-alpha, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Ralpha(+) lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Ralpha(+) lymphocytes, we tested its cytolytic effect on IL-21Ralpha(+) EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Ralpha agonist that co-opts IL-21Ralpha-dependent signaling in a manner permissive for targeted cancer immunotherapy.

Induction of Cardiac Angiogenesis Requires Killer Cell Lectin-Like Receptor 1 and α4β7 Integrin Expression by NK Cells

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2–activated NK cells promoted TNF-α–stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2–activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin–specific binding. In this study, we show that IL-2–activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.

Interleukin-2 enhances angiogenesis and preserves cardiac function following myocardial infarction.

We previously demonstrated that injection of IL-2-activated natural killer (NK) cells contribute to vascular remodeling via a4b7 integrin and killer cell lectin-like receptor (KLRG) 1 and promote cardiac repair following myocardial infarction (MI). The aim of the present study is to test the hypothesis that injection of recombinant human interleukin (rhIL)-2 improves angiogenesis and preserves heart function after MI. A single IV injection of rhIL-2 two days following MI improved by 27.7% the left ventricular (LV) fractional shortening of immune competent (C57Bl6) mice, but had no effect on cardiac function of immune-deficient (NOD-SCID IL2Rγnull) mice. Immunohistochemical analysis of C57Bl6 cross sections of heart revealed that collagen deposition was reduced by 23.1% and that capillary density was enhanced in the scar area and the border zone of the infarct respectively by 22.4% and 33.6% following rhIL-2 injection. In addition, rhIL-2 enhanced 1.6-fold the in vivo endothelial cell proliferation index and 1.8-fold the number of NK cell infiltrating the infarcted heart, but had no effect on the number of cardiac CD4 and CD8 cells. In vitro, rhIL-2 activated NK cells enhanced cardiac endothelial cell proliferation by 17.2%. Here we show that a single IV injection of rhIL-2 positively impacted cardiac function by improving angiogenesis through a process involving NK cells.

The carboxylation efficiency of the vitamin K-dependent clotting factors: studies with factor IX.

Summary.  Haemophilia B is characterized by a deficiency of the γ‐carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach (Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K‐dependent proteins in vitro, to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT‐FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is γ‐carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.