Diminished vasculogenesis under inflammatory conditions is mediated by Activin A.

Severe inflammatory stress often leads to vessel rarefaction and fibrosis, resulting in limited tissue recovery. However, signaling pathways mediating these processes are not completely understood. Patients with ischemic and inflammatory conditions have increased systemic Activin A level, which frequently correlates with the severity of pathology. Yet, Activin A's contribution to disease progression, specifically to vascular homeostasis and remodeling, is not well defined. This study investigated vasculogenesis in an inflammatory environment with an emphasis on Activin A's role. Exposure of endothelial cells (EC) and perivascular cells (adipose stromal cells, ASC) to inflammatory stimuli (represented by blood mononuclear cells from healthy donors activated with lipopolysaccharide, aPBMC) dramatically decreased EC tubulogenesis or caused vessel rarefaction compared to control co-cultures, concurrent with increased Activin A secretion. Both EC and ASC upregulated Inhibin Ba mRNA and Activin A secretion in response to aPBMC or their secretome. We identified TNFα (in EC) and IL-1ß (in EC and ASC) as the exclusive inflammatory factors, present in aPBMC secretome, responsible for induction of Activin A. Similar to ASC, brain and placental pericytes upregulated Activin A in response to aPBMC and IL-1ß, but not TNFα. Both these cytokines individually diminished EC tubulogenesis. Blocking Activin A with neutralizing IgG mitigated detrimental effects of aPBMC or TNFα/IL-1ß on tubulogenesis in vitro and vessel formation in vivo. This study delineates the signaling pathway through which inflammatory cells have a detrimental effect on vessel formation and homeostasis, and highlights the central role of Activin A in this process. Transitory interference with Activin A during early phases of inflammatory or ischemic insult, with neutralizing antibodies or scavengers, may benefit vasculature preservation and overall tissue recovery.

Notch Pathways Regulate Expression of Angiostatic Factor Activin A in Endothelial-Pericyte-Like Mesenchymal Stromal Cell Interactions.

Vascularization of ischemic and fabricated tissues is essential for successful tissue repair and replacement therapies. Endothelial cells (ECs) and mesenchymal stem/stromal cells (MSCs) in close proximity spontaneously organize into vessels after coimplantation in semisolid matrices. Thus, local injection of EC mixed with MSC may facilitate tissue (re)vascularization. The organization of these cells into vessels is accompanied by induction of a key regulator of vasculogenesis, activin A, in MSC through juxtacrine pathway. Mechanisms regulating activin A expression are poorly understood; therefore, the contributions of notch signaling pathways were evaluated in EC-adipose mesenchymal stromal cells (ASC) cocultures. Disruption of notch signaling in EC + ASC cocultures with a γ-secretase inhibitor, DAPT, completely abrogated both activin A induction and production, depending on the stage of vasculogenesis. While DAPT stimulated EC proliferation concurrent with increased secretion of vasculogenic factors, it also prevented the crucial transition of ASC from progenitor to smooth muscle cell phenotype, collectively resulting in ineffective tubulogenesis. Silencing Notch2 in ASC abolished activin A production in cocultures, but resulted in normal ASC maturation. In contrast, silencing Notch3 in ASC led to autonomous upregulation of mural cell markers, and intercellular contact with EC further enhanced upregulation of these markers, concurrent with amplified activin A secretion. Strong induction of activin A expression was achieved by exposing ASC to immobilized notch ligand jagged1, whereas jagged1 IgG, added to EC + ASC incubation media, prevented activin A expression. Overall, this study revealed that EC control activin A expression in ASC through trans juxtacrine notch signaling pathways, and uninterrupted notch signaling is required for activin A production, although signaling through Notch2 and Notch3 produce opposing effects.

Development of a student-driven undergraduate program in regenerative medicine.

As it begins to enter the clinic, regenerative medicine has the potential to revolutionize healthcare. Although there exists a growing need for individuals well-versed in the practice of regenerative medicine, few undergraduate institutions offer opportunities to learn about the topic. This article highlights the conception of two novel undergraduate courses in regenerative medicine developed through collaboration between students and faculty at our University to fill this void in the undergraduate curriculum. Lectures from scientists, healthcare professionals, regulatory experts and biotechnology leaders introduced students to regenerative medicine research and the translational process, and a certificate program incorporating relevant coursework and research experience is in development. This pipeline will guide promising undergraduate students to the field of regenerative medicine.

Regenerative medicine is a new medical discipline that aims to restore diseased or damaged tissue back to a healthy state. Stem cells, gene therapies and other regenerative approaches are now being used to treat patients, and, as a result, the field has recently entered the public eye. To implement these cutting-edge therapies, a well-trained workforce is required; however, regenerative medicine education, especially at the undergraduate level, is currently lacking. Faculty and students at our University worked together to address this issue by creating educational offerings that expose undergraduates to the work being done in the field, and opening opportunities for help them to engage in regenerative medicine-related research. Expanded utilization of this approach will encourage talented undergraduates to contribute to the development of safe, effective regenerative therapies.

Senescence-associated hyper-activation to inflammatory stimuli in vitro.

Aging is associated with an increased susceptibility to adverse inflammatory conditions such as sepsis and cytokine storm. We hypothesized that senescent cells (SnCs) play a central role in this age-associated pathology in part due to their expression of the senescence-associated secretory phenotype (SASP), which may prime SnCs to inflammatory stimulation. To test this hypothesis, we examined the expression of various inflammatory cytokines and chemokines at the levels of gene transcription and protein production in various SnCs in vitro in response to lipopolysaccharide (LPS), interleukin-1ß (IL1ß), and tumor necrosis factor α (TNFα) stimulation. We found that SnCs not only expressed higher basal levels of various inflammatory cytokines and chemokines as a manifestation of the SASP, but more importantly exhibited hyper-activation of the induction of a variety of inflammatory mediators in response to LPS, IL1ß and TNFα stimulation as compared with non-SnCs. This senescence-associated hyper-activation is likely mediated in part via the p38MAPK (p38) and NFκB pathways because LPS stimulation elicited significantly higher levels of p38 phosphorylation and NFκB p65 nuclear translation in SnCs when compared to their non-senescent counterparts and inhibition of these pathways with losmapimod (a p38 specific inhibitor) and BMS-345541 (a selective NFκB inhibitor) attenuated LPS-induced expression of IL6, TNFα, CCL5, and IL1ß mRNA in SnCs. These findings suggest that SnCs may play an important role in the age-related increases in the susceptibility to developing an exacerbated inflammatory response and highlight the potential to use senotherapeutics to ameliorate the severity of various devastating inflammatory conditions in the elderly.

Adipose stem cell secretome markedly improves rodent heart and human induced pluripotent stem cell-derived cardiomyocyte recovery from cardioplegic transport solution exposure.

Heart transplantation is a life-saving therapy for end-stage organ failure. Organ deterioration during transportation limits storage to 4 hours, limiting hearts available. Approaches ameliorating organ damage could increase the number of hearts acceptable for transplantation. Prior studies show that adipose-derived stem/stromal cell secretome (ASC-S) rescues tissues from postischemic damage in vivo. This study tested whether ASC-S preserved the function of mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iCM) exposed to organ transportation and transplantation conditions. Hearts were subjected to cold University of Wisconsin (UW) cardioplegic solution ± ASC-S for 6 hours followed by analysis using the Langendorff technique. In parallel, the effects of ASC-S on the recovery of iCM from UW solution were examined when provided either during or after cold cardioplegia. Exposure of hearts and iCM to UW deteriorated contractile activity and caused cell apoptosis, worsening in iCM as a function of exposure time; these were ameliorated by augmenting with ASC-S. Silencing of superoxide dismutase 3 and catalase expression prior to secretome generation compromised the ASC-S cardiomyocyte-protective effects. In this study, a novel in vitro iCM model was developed to complement a rodent heart model in assessing efficacy of approaches to improve cardiac preservation. ASC-S displays strong cardioprotective activity on iCM either with or following cold cardioplegia. This effect is associated with ASC-S-mediated cellular clearance of reactive oxygen species. The effect of ASC-S on the temporal recovery of iCM function supports the possibility of lengthening heart storage by augmenting cardioplegic transport solution with ASC-S, expanding the pool of hearts for transplantation.

Therapeutic Use of Adipose-Derived Stromal Cells in a Murine Model of Acute Pancreatitis.

BACKGROUND: No specific therapy exists for acute pancreatitis (AP), and current treatment remains entirely supportive. Adipose stem cells (ASCs) have significant immunomodulatory and regenerative activities. We hypothesized that systemic administration of ASCs would mitigate inflammation in AP. METHODS: AP was induced in mice by 6 hourly intraperitoneal injections of cerulein. Twenty-four hours after AP induction, mice were randomized into four systemic treatment groups: sham group (no acute pancreatitis), vehicle, human ASCs, and human ASC-conditioned media. Mice were sacrificed at 48 h, and blood and organs were collected and analyzed. Pancreatic injury was quantified histologically using a published score (edema, inflammation, and necrosis). Pancreatic inflammation was also studied by immunohistochemistry and PCR. RESULTS: When using IV infusion of Hoechst-labeled ASCs, ASCs were found to localize to inflamed tissues: lungs and pancreas. Mice treated with ASCs had less severe AP, as shown by a significantly decreased histopathology score (edema, inflammation, and necrosis) (p = 0.001). ASCs infusion polarized pancreatic macrophages toward an anti-inflammatory M2 phenotype. ASC-conditioned media reduced pancreatic inflammation similarly to ASCs only, highlighting the importance of ASCs secreted factors in modulating inflammation. CONCLUSION: Intravenous delivery of human ASCs markedly reduces pancreatic inflammation in a murine model of AP ASCs which represent an effective therapy for AP.

Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia.

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS-exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856-867.

Hypoxia-induced activin A diminishes endothelial cell vasculogenic activity.

Acute ischaemia causes a significant loss of blood vessels leading to deterioration of organ function. Multiple ischaemic conditions are associated with up-regulation of activin A, but its effect on endothelial cells (EC) in the context of hypoxia is understudied. This study evaluated the role of activin A in vasculogenesis in hypoxia. An in vitro vasculogenesis model, in which EC were cocultured with adipose stromal cells (ASC), was used. Incubation of cocultures at 0.5% oxygen led to decrease in EC survival and vessel density. Hypoxia up-regulated inhibin BA (monomer of activin A) mRNA by 4.5-fold and activin A accumulation in EC-conditioned media by 10-fold, but down-regulated activin A inhibitor follistatin by twofold. Inhibin BA expression was also increased in human EC injected into ischaemic mouse muscles. Activin A secretion was positively modulated by hypoxia mimetics dimethyloxalylglycine and desferrioxamine. Silencing HIF1α or HIF2α expression decreased activin A secretion in EC exposed to hypoxia. Introduction of activin A to cocultures decreased EC number and vascular density by 40%; conversely, blockade of activin A expression in EC or its activity improved vasculogenesis in hypoxia. Activin A affected EC survival directly and by modulating ASC paracrine activity leading to diminished ability of the ASC secretome to support EC survival and vasculogenesis. In conclusion, hypoxia up-regulates EC secretion of activin A, which, by affecting both EC and adjacent mesenchymal cells, creates a micro-environment unfavourable for vasculogenesis. This finding suggests that blockade of activin A signalling in ischaemic tissue may improve preservation of the affected tissue.

Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells.

Background: The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives: We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods: SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results: The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions: By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence: 5.

Human adipose stromal cell therapy improves survival and reduces renal inflammation and capillary rarefaction in acute kidney injury.

Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short-term recovery and long-term functional preservation post-AKI. Human adipose stromal cells (hASCs) possess pro-angiogenic and anti-inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC-treated rats had significantly greater survival compared to vehicle-treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post-AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC-treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle-treated rats as well as reduced accumulation of interstitial alpha-smooth muscle actin-positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R-induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.

Adipose stromal cells differentiation toward smooth muscle cell phenotype diminishes their vasculogenic activity due to induction of activin A secretion.

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell-contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC-lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor ß1 (TGFß1 ) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre-differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre-differentiated ASCs were used. Pre-differentiated ASCs had an inferior mitogenic response to EC-produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co-culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFß1 induced secretion of activin A. Augmenting co-culture incubation media with anti-activin A IgG restored the ability of pre-differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFß1 or activin A-induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co-injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.

Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia.

Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double (p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent therapeutic cocktail for patients with CLI, many of whom are at risk for amputation of the affected limb.

Adipose Stromal Cell Contact with Endothelial Cells Results in Loss of Complementary Vasculogenic Activity Mediated by Induction of Activin A.

Adipose stem/stromal cells (ASCs) after isolation produce numerous angiogenic growth factors. This justifies their use to promote angiogenesis per transplantation. In parallel, local coimplantation of ASC with endothelial cells (ECs) leading to formation of functional vessels by the donor cells suggests the existence of a mechanism responsible for fine-tuning ASC paracrine activity essential for vasculogenesis. As expected, conditioned media (CM) from ASC promoted ECs survival, proliferation, migration, and vasculogenesis. In contrast, media from EC-ASC cocultures had neutral effects upon EC responses. Media from cocultures exhibited lower levels of vascular endothelial growth factor (VEGF), hepatic growth factor, angiopoietin-1, and stromal cell-derived factor-1 compared with those in ASC CM. Activin A was induced in ASC in response to EC exposure and was responsible for overall antivasculogenic activity of EC-ASC CM. Except for VEGF, activin A diminished secretion of all tested factors by ASC. Activin A mediated induction of VEGF expression in ASC, but also upregulated expression of VEGF scavenger receptor FLT-1 in EC in EC-ASC cocultures. Blocking the FLT-1 expression in EC led to an increase in VEGF concentration in CM. In vitro pre-exposure of ASC to low number of EC before subcutaneous coimplantation with EC resulted in decrease in vessel density in the implants. In vitro tests suggested that activin A was partially responsible for this diminished ASC activity. This study shows that neovessel formation is associated with induction of activin A expression in ASC; this factor, by affecting the bioactivity of both ASC and EC, directs the crosstalk between these complementary cell types to establish stable vessels.

Conditioned media from adipose stromal cells limit lipopolysaccharide-induced lung injury, endothelial hyperpermeability and apoptosis.

BACKGROUND: Acute Respiratory Distress Syndrome (ARDS) is a condition that contributes to morbidity and mortality of critically ill patients. We investigated whether factors secreted by adipose stromal cells (ASC) into conditioned media (ASC-CM) will effectively decrease lung injury in the model of lipopolysaccharide (LPS)-induced ARDS. METHODS: To assess the effect of ASC-CM on ARDS indices, intravenous delivery of ASC and ASC-CM to C57Bl/6 mice was carried out 4 h after LPS oropharyngeal aspiration; Evans Blue Dye (EBD) was injected intravenously 1 h prior to animal sacrifice (48 h post-LPS). Lungs were either fixed for histopathology, or used to extract bronchoalveolar lavage fluid (BALF) or EBD. To assess the effect of ASC-CM on endothelial barrier function and apoptosis, human pulmonary artery endothelial cells were treated with ASC-CM for 48-72 h. RESULTS: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6. White Blood Cells (WBC) from BALF of LPS-challenged mice receiving ASC-CM had decreased reactive oxygen species (ROS) generation compared to WBC from LPS-challenged mice receiving control media injection. Treatment of pulmonary endothelial monolayers with ASC-CM significantly suppressed H2O2-induced leakage of FITC dextran and changes in transendothelial resistance, as well as gap formation in endothelial monolayer. ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3. ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation. CONCLUSIONS: Factors secreted by ASC efficiently reduce ARDS indices, endothelial barrier hyperpermeability, and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.

Human adipose-derived stem cells ameliorate cigarette smoke-induced murine myelosuppression via secretion of TSG-6.

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.

Intravenous xenogeneic transplantation of human adipose-derived stem cells improves left ventricular function and microvascular integrity in swine myocardial infarction model.

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 × 10(7) ASCs), or the divided dose group (DD) (n = 7, 5 × 10(7) 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.

Adipose stromal cells differentiate along a smooth muscle lineage pathway upon endothelial cell contact via induction of activin A.

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-ß1 in conditioned media. This was attributed to induction of activin A expression in ASC on contact with EC. Although transforming growth factor-ß 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-ß was able to induce activin A expression in ASC, in cocultures this induction was transforming growth factor-ß 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.

Human adipose-derived stromal/stem cells protect against STZ-induced hyperglycemia: analysis of hASC-derived paracrine effectors.

Adipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved ß cell mass, and increased ß cell proliferation in streptozotocin-treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC-derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP-1) to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against ß cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects.

Regenerative therapeutic potential of adipose stromal cells in early stage diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved "b" wave amplitude (as measured by electroretinogram) within 1-3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.