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Rationale: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. Objective: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. Methods and Results: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2 −/y , Akita (type 1 diabetes mellitus), and ACE2 −/y -Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2 −/y -Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2 −/y -Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. Conclusions: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2 −/y -Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

A healthy microbiome is central to good nutrition and efficient metabolism both of which can be altered in diabetes. Disruption of gut-vascular integrity may lead to translocation of microbial antigens and bacteria into the circulation. Bone marrow-derived circulating angiogenic cells (CACs) are essential to maintain vascular integrity. We tested the hypothesis that angiotensin-converting enzyme 2 (ACE2), the key enzyme in the vasoprotective axis of renin-angiotensin system (RAS), acts to regulate the infiltration of bone marrow-derived CACs in the gut and to maintain gut-vascular integrity in diabetes and could influence the systemic translocation of pathogenic antigens and bacteria from the gut. Diabetic ACE2 knockout (ACE2-/y)/C57BL/6-Ins2WT/C96Y (Akita) mice were examined at 9 months after the onset of diabetes and compared to age-matched controls. Beta diversity analysis of the gut microbiome revealed a distinct bacterial profile among the different genotypes (n=9-17 per group, p=0.002). The fecal samples from ACE2-/y-Akita group compared to Akita group were enriched with a great diversity of bacterial taxa that have been reported to contribute to the pathogenesis of diabetes and showed activation of peptidoglycan biosynthesis pathways by metatranscriptome analysis (n=4 per group, p=0.005). Flow cytometry analysis showed that loss of ACE2 led to reduced infiltration of bone marrow-derived CACs (CD45+Flk-1+CD31+) into the gut (n=8-14 per group, p Disclosure Y. Duan: None. D. Feng: None. E. Beli: None. S. Li Calzi: None. A.F. Longhini: None. R. Lamendella: None. J.R. Wright: None. T.A. Markel: None. A.G. Obukhov: None. M.B. Grant: None.

Objectives The potential for beneficial effects of adipose-derived stem cells (ASCs) on myocardial perfusion and left ventricular dysfunction in myocardial ischemia (MI) has not been tested following intravenous delivery. Methods Surviving pigs following induction of MI were randomly assigned to 1 of 3 different groups: the placebo group (n = 7), the single bolus group (SB) (n = 7, 15 × 107 ASCs), or the divided dose group (DD) (n = 7, 5 × 107 ASCs/day for three consecutive days). Myocardial perfusion defect area and coronary flow reserve (CFR) were compared during the 28-day follow-up. Also, serial changes in the absolute number of circulating CD4+T and CD8+T cells were measured. Results The increases in ejection fraction were significantly greater in both the SB and the DD groups compared to the placebo group (5.4 ± 0.9%, 3.7 ± 0.7%, and −0.4 ± 0.6%, respectively), and the decrease in the perfusion defect area was significantly greater in the SB group than the placebo group (−36.3 ± 1.8 and −11.5 ± 2.8). CFR increased to a greater degree in the SB and the DD groups than in the placebo group (0.9 ± 0.2, 0.8 ± 0.1, and 0.2 ± 0.2, respectively). The circulating number of CD8+T cells was significantly greater in the SB and DD groups than the placebo group at day 7 (3,687 ± 317/µL, 3,454 ± 787/µL, and 1,928 ± 457/µL, respectively). The numbers of small vessels were significantly greater in the SB and the DD groups than the placebo group in the peri-infarct area. Conclusions Both intravenous SB and DD delivery of ASCs are effective modalities for the treatment of MI in swine. Intravenous delivery of ASCs, with its immunomodulatory and angiogenic effects, is an attractive noninvasive approach for myocardial rescue. © 2014 Wiley Periodicals, Inc.

Objective: Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays. Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion: Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression. Stem Cells 2015;33:468–478

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2–/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2–/y-Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage–c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2–/y-Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy.

The interaction between human endothelial (EC) and adipose mesenchymal stem/stromal cells (ASC) leads to EC reorganization into stable vascular structures in vitro and functional vessels in vivo and ASC maturation from progenitor to smooth muscle/pericyte phenotype. We have previously shown that juxtacrine EC-ASC interactions induce in ASC to express activin A, which, by affecting both EC and ASC bioactivities, plays a central role in vasculogenesis. Here, a co-culture model of human cord blood-derived EC and human ASC was used to study the role of Notch2 and Notch3 in juxtacrine mechanism-mediated regulation of activin A expression in ASC. Blockade of Notch signaling by the γ-secretase inhibitor DAPT, between days 1-3 following heterocellular contact, prevented induction of activin A secretion, and DAPT added to co-cultures after establishment of activin A secretion almost completely abrogated its production, suggesting the need for uninterrupted Notch signaling to enforce continued activin A production. In parallel, DAPT reduced ASC differentiation towards the mural phenotype, and increased EC proliferation, causing inefficient vasculogenesis. Silencing Notch2 expression in ASC prevented activin A production by ASC when in co-culture with EC, whereas silencing Notch3 expression amplified its secretion. Dual silencing experiments revealed that silencing Notch2 was dominant, eliminating the effect of silencing Notch3. In parallel, Notch2 was not involved in mediating differentiation of ASC towards a smooth muscle/pericyte fate, whereas silencing of Notch3 led to an increase in baseline expression of markers of smooth muscle phenotype, which was further enhanced on contact with EC. Overall, Notch3 inhibits both expression of activin A and smooth muscle/pericyte differentiation program. Furthermore, these data suggest that activin A is induced exclusively through trans signaling mechanisms, whereas mural differentiation program is also regulated through cis pathways.

Objectives To examine the comparative fate of adipose-derived stem cells (ASCs) as well as their impact on coronary microcirculation following either retrograde coronary venous or arterial delivery.

Abdominal aortic aneurysm (AAA) is a potentially lethal disease associated with immune activation-induced aortic degradation. We hypothesized that xenotransplantation of human adipose-derived stem cells (hADSCs) would reduce aortic inflammation and attenuate expansion in a murine AAA model. Modulatory effects of ADSCs on immune cell subtypes associated with AAA progression were investigated using human peripheral blood mononuclear cells (hPBMNCs) cocultured with ADSCs. Murine AAA was induced through elastase application to the abdominal aorta in C57BL/6 mice. ADSCs were administered intravenously, and aortic changes were determined by ultrasonography and videomicrometry. Circulating monocytes, aortic neutrophils, CD28−T cells, FoxP3+regulatory T cells (Tregs), and CD206+M2 macrophages were assessed at multiple terminal time points. In vitro, ADSCs induced M2 macrophage and Treg phenotypes while inhibiting neutrophil transmigration and lymphocyte activation without cellular contact. Intravenous ADSC delivery reduced aneurysmal expansion starting from day 4 [from baseline: 54.8% (saline) vs. 16.9% (ADSCs), n = 10 at baseline, n = 4 at day 4, p < 0.001], and the therapeutic effect persists through day 14 (from baseline: 64.1% saline vs. 24.6% ADSCs, n = 4, p < 0.01). ADSC administration increased aortic Tregs by 20-fold ( n = 5, p < 0.01), while decreasing CD4+CD28−(-28%), CD8+CD28−T cells (-61%), and Ly6G/C+neutrophils (-43%, n = 5, p < 0.05). Circulating CD115+CXCR1−LY6C+-activated monocytes decreased in the ADSC-treated group by day 7 (-60%, n = 10, p < 0.05), paralleled by an increase in aortic CD206+M2 macrophages by 2.4-fold ( n = 5, p < 0.05). Intravenously injected ADSCs transiently engrafted in the lung on day 1 without aortic engraftment at any time point. In conclusion, ADSCs exhibit pleiotropic immunomodulatory effects in vitro as well as in vivo during the development of AAA. The temporal evolution of these effects systemically as well as in aortic tissue suggests that ADSCs induce a sequence of anti-inflammatory cellular events mediated by paracrine factors, which leads to amelioration of AAA progression.

Introduction: Abdominal aortic aneurysm (AAA) formation is characterized by activation of innate and adaptive immune responses that initiate aortic wall degradation. We hypothesize that human adipose stromal cells (ASC) favorably modulate these immune responses through paracrine mechanisms and attenuate AAA expansion in a murine elastase model. Methods: AAA was induced in C57BL/6 mice by topical treatment of the abdominal aorta with elastase. Human ASC or saline was injected i.v. within 4 hours of elastase treatment. The aortic diameter was measured by ultrasound and video micrometry. Monocytes/macrophages and lymphocyte sub-populations cells were quantified by flow cytometry. Regulatory T cells (Treg) and M2 macrophages were quantified by immunohistochemical (IHC) staining. ASC were labeled with Hoechst and their distributions in the lung and aorta were imaged fluorescent microscopy and confirmed by real-time PCR for human-specific Alu sequence. Results: ASC significantly suppressed AAA expansion at 14 days (n=10/group, p Conclusion: Systemic injection of ASC significantly suppresses AAA formation by inducing anti-inflammatory immune cell phenotypes. These effects are mediated by release of paracrine factors and suggest that further characterization of the ASC secretome may lead to more potent recombinant protein therapies for AAA.

Rationale: Adipose stromal cells (ASC) are therapeutically potent progenitor cells that possess properties of pericytes. In vivo, ASC in combination with endothelial cells (EC) establish functional multilayer vessels, in which ASC form the outer vessel layer and differentiate into mural cells. Objective: To identify factors responsible for ASC differentiation toward the smooth muscle cell phenotype via interaction with EC. Methods and Results: An in vitro model of EC cocultivation with ASC was used, in which EC organized into vascular cords, accompanied by ASC migration toward EC and upregulation of α-smooth muscle actin, SM22α, and calponin expression. Conditioned media from EC–ASC, but not from EC cultures, induced smooth muscle cell protein expression in ASC monocultures. EC–ASC cocultivation induced marked accumulation of activin A but not transforming growth factor-β1 in conditioned media. This was attributed to induction of activin A expression in ASC on contact with EC. Although transforming growth factor-β and activin A were individually sufficient to initiate expression of smooth muscle cell antigens in ASC, only activin A IgG blocked the effect of EC–ASC conditioned media. Although transforming growth factor-β was able to induce activin A expression in ASC, in cocultures this induction was transforming growth factor-β independent. In EC–ASC cocultures, activin A IgG or ALK4/5/7 receptor inhibitors blocked expression of α-smooth muscle actin in ASC in the absence of direct EC-cord contact, but this inhibition was circumvented in ASC by direct EC contact. Conclusions: EC initiate a smooth muscle cell differentiation program in adjacent ASC and propagate this differentiation in distant ASC by induction of activin A expression.

Rationale: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. Objectives: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. Methods: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. Measurements and Main Results: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. Conclusions: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.

Transplantation of pluripotent adipose stem/stromal cells (ASC) alleviates tissue damage and improves functional deficits in both stroke and cardiovascular disease animal models. Recent studies indicate that the primary mechanism of ASC-induced repair may not be directly related to tissue regeneration through differentiation, but rather through paracrine mechanisms provided by secreted pro-survival and repair-inducing trophic factors. In this study, we have found that ASC-conditioned medium (ASC-CM) potently protected cerebellar granule neurons (CGN) from apoptosis induced by serum and potassium deprivation. Neural cell protection was mostly attributable to activated caspase-3 and Akt-mediated neuroprotective pathway signaling. Specific neutralization of neurotrophic factor activity demonstrated that serum and potassium deprivation-induced Akt-mediated neuroprotection and caspase-3-dependent apoptosis were mainly modulated by IGF-1. These data suggest that of the many neuroprotective factors secreted by ASC, IGF-1 is the major factor that mediates protection against serum and potassium deprivation-induced CGN apoptosis. This study establishes a mechanistic basis supporting the therapeutic application of ASC for neurological disorders, specifically through paracrine support provided by trophic factor secretion.

The delivery of factors secreted by adipose stromal cells (ASC) to the brain may be a viable neuroprotective therapeutic option. In this study, we investigated the bioactivity of ASC-conditioned medium (ASC-CM) in glutamate-induced neurotoxicity and found that the ASC-CM significantly blocked glutamate neurotoxicity. We identified the brain-derived neurotrophic factor (BDNF) in the ASC-CM by using Western blot and demonstrated that this activity was critical for the neuroprotective effect of ASC-CM in excytotoxicity models. Furthermore, inactivating BDNF also attenuated the suppression by ASC-CM of glutamate-induced caspase-3 activity, but not p38 phosphorylation. These findings suggest that among ASC secrete a potent combination of factors, BDNF play a major role in neuroprotection against excytotoxicity.

Background Paracrine stimulation of endogenous repair, rather than direct tissue regeneration, is increasingly accepted as a major mode of therapeutic stem and progenitor cell action; yet, this principle has not been fully established in vivo . Adipose-derived stem cells (ASCs) secrete many factors and promote reperfusion and tissue repair in ischemia models. RNA interference was used to silence the expression of the abundant protein, hepatocyte growth factor (HGF), to determine its contribution to ASC potency in vivo . Methods and Results Dual-cassette lentiviral vectors, expressing GFP and either a small hairpin RNA (shRNA) specific for HGF mRNA (shHGF) or a control sequence (shCtrl), were used to stably transduce ASCs (ASC-shHGF or ASC-shCtrl). ASC-shHGF secreted 5-fold less HGF, which resulted in a reduced ability of these cells to promote survival, proliferation and migration of mature and progenitor endothelial cells in vitro ( p p p ASC-shHGF>saline) ( p + cells in ischemic limbs at 5 d after infusion, indicating similar homing potentials, 3-fold fewer ASC-shHGF were present in ischemic tissues at 15 d compared to ASC-shCtrl ( p p Conclusions These results indicate that secretion of HGF is critically important for ASC potency. In addition to promoting endogenous repair, the data suggest that an important effect of HGF is autocrine promotion of ASC survival in ischemic tissue. Enhanced donor cell survival is an important goal for increasing the efficacy of cell therapy.