Hongyan Lu

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 &agr;-smooth muscle actin, SM22&agr;, 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-&bgr;1 in conditioned media. This was attributed to induction of activin A expression in ASC on contact with EC. Although transforming growth factor-&bgr; 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-&bgr; was able to induce activin A expression in ASC, in cocultures this induction was transforming growth factor-&bgr; independent. In EC–ASC cocultures, activin A IgG or ALK4/5/7 receptor inhibitors blocked expression of &agr;-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.

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. Stem Cells 2015;33:3039–3051

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.

Pulmonary Retention of Adipose Stromal Cells following Intravenous Delivery is Markedly Altered in the Presence of ARDS

Transplantation of mesenchymal stromal cells (MSCs) has been shown to effectively prevent lung injury in several preclinical models of acute respiratory distress syndrome (ARDS). Since MSC therapy is tested in clinical trials for ARDS, there is an increased need to define the dynamics of cell trafficking and organ-specific accumulation. We examined how the presence of ARDS changes retention and organ-specific distribution of intravenously delivered MSCs isolated from subcutaneous adipose tissue [adipose-derived stem cells (ADSCs)]. This type of cell therapy was previously shown to ameliorate ARDS pathology. ARDS was triggered by lipopolysaccharide (LPS) aspiration, 4 h after which 300,000 murine CRE+ ADSCs were delivered intravenously. The distribution of ADSCs in the lungs and other organs was assessed by real-time polymerase chain reaction (PCR) of genomic DNA. As anticipated, the majority of delivered ADSCs accumulated in the lungs of both control and LPS-challenged mice, with minor amounts distributed to the liver, kidney, spleen, heart, and brain. Interestingly, within 2 h following ADSC administration, LPS-challenged lungs retained significantly lower levels of ADSCs compared to control lungs (~7% vs. 15% of the original dose, respectively), whereas the liver, kidney, spleen, and brain of ARDS-affected animals retained significantly higher numbers of ADSCs compared to control animals. In contrast, 48 h later, only LPS-challenged lungs continued to retain ADSCs (~3% of the original dose), whereas the lungs of control animals and nonpulmonary organs in either control or ARDS mice had no detectable levels of ADSCs. Our data suggest that the pulmonary microenvironment during ARDS may lessen the pulmonary capillary occlusion by MSCs immediately following cell delivery while facilitating pulmonary retention of the cells.

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

EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury

Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.

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.