Paracrine Functions Research Articles

Epicardial atrial fat and left atrium remodeling

Increasing data have reported a potent link between epicardial atrial fat (EAF) and atrial fibrillation (AF) that could contribute to atrial substrate remodeling through its paracrine function. High burden of AF is associated with progressive left atrium (LA) structural remodeling but little is known about the relationship between LA reverse remodeling (LARR) and EAF thickness evolution according to AF burden. The objective of the study was to evaluate EAF thickness using 2 consecutive CT scan in a cohort of patients with recurrent AF after ablation and to analyze its relation with LA volume and LARR. We included 90 patientswho had a first AF ablation and a redo procedure along with two computed tomographies (CT). EAF thickness and LARR defined as a 15% reduction of LA volume were measured on 2 consecutive CT. The patients were divided into two groups–those with LARR (LARR +) and those without (LARR-). LARR occurred in 23% (21) of the cohort despite AF recurrence in both group. There was no significant difference in terms of age, BMI, cardiovascular risk factors between the two groups. LA volume was the only multivariate predictor of an important EAF thickness (HR 1.01; 95%CI 1.001–1.026; P = 0.04). BMI was not associated to EAF. Hypertension and EAF thickness (HR 5.71; 95%CI 1.22–26.75; P = 0.009; HR 1.41; 95%CI 1.09–1.84; P = 0.009 respectively) were independent predictive factors of LA enlargement whereas glomerular filtration rate was protective (HR 0.95; 95%CI 0.92–0.99; P = 0.010). We could not identify LARR predictor except ACEI/ARB treatment (HR 2.78; 95%; CI 1.023–7.597; P = 0.044). No significant decrease of EAF could be observed in LARR+ and LARR- groups. LA volume was the sole independent predictor of EAF thickness. Hypertension, EAF thickness were associated with LA enlargement. ACEI/ARB was predictive of LARR. No significant decrease in EAF thickness was associated with reverse remodeling after AF ablation.

A dynamic matrix potentiates mesenchymal stromal cell paracrine function via an effective mechanical dose.

The paracrine function of mesenchymal stromal cells (MSCs) contributes a lot to tissue development, and it is regulated by various physical factors. Moreover, the extracellular matrix (ECM) of MSCs is dynamic, and its remodeling is always occurring. In particular, stiffness changes are prevalent. Accordingly, ECM stiffness changes may affect the paracrine function of MSCs, which has not been investigated much. In this study, for the first time, alginate hydrogels with different stiffening times were used to assess the influence of dynamic ECM stiffness changes on the paracrine function of MSCs. It was found that a stiffer matrix (14.72 ± 1.44 kPa) under static conditions without any additional treatment could significantly potentiate the paracrine function of MSCs compared to a soft matrix (2.44 ± 0.23 kPa). Furthermore, this promotion was regulated by the activation of Yes-associated protein (YAP), which was caused by the polymerization of F-actin. Intriguingly, in a dynamic system, the MSC-encapsulating matrix that stiffened on the third day had stronger YAP activation than the Static-Stiff matrix. However, this activation was weakened when MSCs were cultured in a matrix that stiffened on the fifth day. The results show that an increase in ECM mechanical dosing levels would promote the paracrine function of MSCs. Moreover, an effective mechanical dose that can influence the paracrine function of MSCs indeed exists.

Autologous Bone Marrow Mononuclear Cell Transplantation Therapy Improved Symptoms in Patients with Refractory Diabetic Sensorimotor Polyneuropathy via the Mechanisms of Paracrine and Immunomodulation: A Controlled Study

We recently reported that transplantation of autologous bone marrow mononuclear cells (BM-MNCs) may be an effective and promising therapy to treat refractory diabetic sensorimotor polyneuropathy (DSPN) in patients with type 2 diabetes mellitus (T2DM). This study was designed to investigate the potential mechanisms of BM-MNCs therapy, which recruited 60 patients with DSPN, 30 T2DM patients without complications, and 30 healthy control participants. All clinical parameters, the levels of inflammatory markers, and growth factors in the three groups were compared. Patients in DSPN group had higher level of tumor necrosis factor-α (TNF-α) (DSPN vs control, 412.90 ± 64.58 vs 374.81 ± 63.18 pg/mL, P < 0.01) and lower level of vascular endothelial growth factor (VEGF) (DSPN vs control, 140.93 ± 24.78 vs 157.39 ± 25.11 pg/mL, P < 0.01) than those in control group. DSPN group had the highest level of soluble intercellular adhesion molecule-1 (sICAM-1) among three groups (DSPN and DM vs control, 1477.56 ± 228.00 and 1342.17 ± 237.54 vs 1308.00 ± 200.94 ng/mL, P < 0.05). The level of nerve growth factor in the DSPN group was slightly lower than that in the DM group (DSPN vs DM, 3509.11 ± 438.39 vs 3734.87 ± 647.50 pg/mL, P < 0.05). All patients with DSPN received one intramuscular injection of BM-MNCs and clinical follow-ups after the therapy for 2 days, 1, 4, 12, 24, and 48 weeks. Neuropathic symptoms of foot pain, numbness, and weakness were significantly improved within 4 weeks after BM-MNCs injection. Patients with DSPN were divided into the responder (n = 35) and nonresponder groups (n = 19) based on the improvement of nerve conduction velocity at 12 weeks post-transplantation. Compared with nonresponders, responders were younger (57.3 ± 5.2 vs 62.0 ± 4.8, P < 0.01), had a shorter history of diabetes (7.1 ± 2.7 vs 11.2 ± 5.4 years, P < 0.01), and had higher numbers of mobilized CD34+ cells (17.61 ± 2.64 vs 14.79 ± 1.62 ×105/L, P < 0.01) and BM-MNCs (12.05 ± 2.16 vs 9.84 ± 1.53 ×108/L, P < 0.01). The levels of TNF-α and sICAM-1 decreased just after BM-MNCs injection in both groups and slowly reverted to baseline levels. The duration of the downtrend of TNF-α and sICAM-1 in the responder group lasted longer than that in the nonresponder group. Serum level of VEGF in the responder group increased immediately after BM-MNC therapy and reached the highest point after the injection for 12 weeks. On the other hand, VEGF levels in the nonresponder group only increased slightly. Binary logistic regression was performed to evaluate the corresponding prognostic factors for BM-MNCs treatment. The number of applied CD34+ cells and the duration of diabetes were the independent predictors of responding to BM-MNCs therapy. No adverse event associated with the treatment was observed during follow-up observations. These results indicated that BM-MNCs transplantation is an effective and promising therapeutic strategy to treat refractory DSPN. The immune regulation and paracrine function of BM-MNCs may contribute to the improvement of DSPN. Trial registration: Chinese Clinical Trial Registry, ChiCTR-TRC-12002570. URL: http://www.chictr.org.cn/showproj.aspx?proj=6981

Human amniotic mesenchymal stromal cells promote bone regeneration via activating endogenous regeneration.

Rationale: The effectiveness of stem cell based-therapy for bone regeneration has been demonstrated; yet, clinical application of autologous stem cells is still limited by invasive acquisition, long culture processes, and high cost. Besides, it remains controversial whether autologous stem cells could directly participate in tissue repair after differentiation. Thus, increasing allogeneic stem cells have been developed into drugs to indirectly activate endogenous regeneration and induce tissue regeneration. Human amniotic mesenchymal stromal cells (HAMSCs) have been extensively studied, showing multiple regulatory functions, but mechanisms of HAMSCs in promoting bone regeneration are remain unclear.Methods: Proteome profile of HAMSCs and their functions on vascularized bone regeneration were investigated in vitro, while rabbit cranial defect model was used to further detect the effects of bone formation in vivo.Results: HAMSCs secrete many osteogenic, angiogenic, and immunomodulatory cytokines. In vitro, HAMSCs can promote human bone-marrow mesenchymal stromal cells (HBMSCs) migration and osteogenic differentiation; promote the capillary-tube formation of human umbilical vascular endothelial cells (HUVECs), induce HUVECs migration and pro-angiogenic genes expression, and promote M2 macrophage polarization. Further, in vivo studies suggested that transplanted HAMSCs could survive and induce M2 macrophages to secrete bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) in rabbits' skull defects at an early stage, and, in turn, promote more new bone formation.Conclusion: HAMSCs have good biocompatibility and paracrine function to promote bone repair by stimulating endogenous regeneration.

Paracrine Proangiogenic Function of Human Bone Marrow-Derived Mesenchymal Stem Cells Is Not Affected by Chronic Kidney Disease.

Background Cell-based therapies are being developed to meet the need for curative therapy in chronic kidney disease (CKD). Bone marrow- (BM-) derived mesenchymal stromal cells (MSCs) enhance tissue repair and induce neoangiogenesis through paracrine action of secreted proteins and extracellular vesicles (EVs). Administration of allogeneic BM MSCs is less desirable in a patient population likely to require a kidney transplant, but potency of autologous MSCs should be confirmed, given previous indications that CKD-induced dysfunction is present. While the immunomodulatory capacity of CKD BM MSCs has been established, it is unknown whether CKD affects wound healing and angiogenic potential of MSC-derived CM and EVs. Methods MSCs were cultured from BM obtained from kidney transplant recipients (N = 15) or kidney donors (N = 17). Passage 3 BM MSCs and BM MSC-conditioned medium (CM) were used for experiments. EVs were isolated from CM by differential ultracentrifugation. BM MSC differentiation capacity, proliferation, and senescence-associated β-galactosidase activity was assessed. In vitro promigratory and proangiogenic capacity of BM MSC-derived CM and EVs was assessed using an in vitro scratch wound assay and Matrigel angiogenesis assay. Results Healthy and CKD BM MSCs exhibited similar differentiation capacity, proliferation, and senescence-associated β-galactosidase activity. Scratch wound migration was not significantly different between healthy and CKD MSCs (P = 0.18). Healthy and CKD BM MSC-derived CM induced similar tubule formation (P = 0.21). There was also no difference in paracrine regenerative function of EVs (scratch wound: P = 0.6; tubulogenesis: P = 0.46). Conclusions Our results indicate that MSCs have an intrinsic capacity to produce proangiogenic paracrine factors, including EVs, which is not affected by donor health status regarding CKD. This suggests that autologous MSC-based therapy is a viable option in CKD.

Current Assays for the Assessment of Endothelial Progenitor Cell Proliferation, Migration, and Paracrine Functions

Endothelial progenitor cells (EPCs), a subset of bone-marrow derived pluripotent mononuclear cells, have been touted for their prognostic, diagnostic, and therapeutic value in the treatment of diseases of the vasculature. However, the results of the clinical trials of autologous EPC transplantation have been mixed, likely because the EPC function of patients who would benefit from autologous transplantation is compromised by genetic and environmental factors associated with pre-clinical and frank disease states. This finding, combined with an inability to assess the functional state of donor EPCs prior to transplantation, creates a significant barrier to their therapeutic application. Although there is currently substantial literature focusing on diseases that impact EPC function, there are no reviews that collate the obligate functions of EPCs for transplantation application. Across the literature, EPC functional assays assess six known abilities of these cells: pre-terminal differentiation, mobilization, proliferation, homing/migration, paracrine functions, and terminal differentiation. This review provides an overview of the current literature on the subset of functions that are crucial for therapeutic efficacy: proliferation, migration and paracrine functions. In this systematic review of over 400 publications, assays that assess these functions are described, organized and evaluated for application in pre-transplant assessment.

Construction of a dermis-fat composite in vivo: Optimizing heterogeneous acellular dermal matrix with in vitro pretreatment.

Dermis-fat composite tissues have been widely used in plastic and reconstructive surgery and were previously constructed using hydrogel-type scaffolds. The constructs can be used for in vitro cosmetic and pharmaceutical testing but are not mechanically strong enough for in vivo applications. In this study, we used heterogeneous (porcine) acellular dermal matrix (PADM) as dermal layer scaffold. PADM was pretreated with the laser micropore technique and then precultured with rat adipose-derived stem cells (rADSCs) in vitro. rADSCs proliferated well on pretreated/unpretreated PADM, showing increased expression of genes associated with inflammatory regulation, proangiogenesis, and stemness, indicating that pretreated/unpretreated PADM both provide a beneficial microenvironment for rADSCs to exert their paracrine function. After in vitro processing, the rADSCs-polyporous PADM and PADM without pretreatments were implanted into the back of rats respectively, followed by adipose tissue transplantation. After implantation, the inflammation induced by pretreated PADM was significantly attenuated and localized compared to the unpretreated group. Moreover, the vascularization was faster, and more adipose tissue was formed in the pretreated group. Sound dermis-fat composite tissue was constructed with sufficient strength, which can potentially be used for actual repair application.

Adipose-Derived Stem Cells from Systemic Sclerosis Patients Maintain Pro-Angiogenic and Antifibrotic Paracrine Effects In Vitro.

Innovative therapies based on autologous adipose-derived stem/stromal cells (ASC) are currently being evaluated for treatment of systemic sclerosis (SSc). Although paracrine angiogenic and antifibrotic effects are considered the predominant mechanisms of ASC therapeutic potential, the impact of SSc on ASC paracrine functions remains controversial. In this study, phenotype, senescence, differentiation potential, and molecular profile were determined in ASC from SSc patients (SSc-ASC) (n = 7) and healthy donors (HD-ASC) (n = 7). ASC were co-cultured in indirect models with dermal fibroblasts (DF) from SSc patients or endothelial cells to assess their pro-angiogenic and antifibrotic paracrine effects. The angiogenic activity of endothelial cells was measured in vitro using tube formation and spheroid assays. DF collagen and alpha smooth muscle actin (αSMA) content were quantified after five days of co-culture with ASC. Differentiation capacity, senescence, and mRNA profiles did not differ significantly between SSc-ASC and HD-ASC. SSc-ASC retained the ability to stimulate angiogenesis through paracrine mechanisms; however, functional assays revealed reduced potential compared to HD-ASC. DF fibrosis markers were significantly decreased after co-culture with SSc-ASC. Together, these results indicate that SSc effects do not significantly compromise the angiogenic and the antifibrotic paracrine properties of ASC, thereby supporting further development of ASC-based autologous therapies for SSc treatment.

Effects of food deprivation on plasma insulin-like growth factor-1 (Igf1) and Igf binding protein (Igfbp) gene transcription in juvenile cabezon (Scorpaenichthys marmoratus)

The growth hormone (GH)/insulin-like growth factor (Igf) endocrine axis regulates somatic growth in the face of changing environmental conditions. In actinopterygian fishes, food availability is a key modulator of the somatotropic axis, with lower food intake generally depressing liver Igf1 release to diminish growth. Igf1 signaling, however, also involves several distinct IGF binding proteins (Igfbps), and the functional roles of many of these Igfbps in affecting growth during shifting food availability remain uncertain. Here, we tested how complete food deprivation (fasting) affected gene transcription for paralogs of all six types of Igfbps in the liver and fast-twitch skeletal muscle of cabezon (Scorpaenichthys marmoratus), a nearshore marine fish important for recreational fisheries in the eastern North Pacific Ocean. Juvenile cabezon were maintained as either fed (6% mass food⋅g fish wet mass−1⋅d−1) or fasted for 14 d. Fasted fish exhibited a lower body condition (K), a depressed mass-specific growth rate (SGR), and reduced plasma concentrations of Igf1. In the liver, fasting reduced the relative abundance of gene transcripts encoding Igfbps igfbp2a and igfbp2b, while significantly elevating mRNA levels for igfbp1a, igfbp1b, igfbp3b, and igfbp4. Fasting also reduced hepatic mRNA levels of GH receptor-1 (ghr1) – but not GH receptor-2 (ghr2) – supporting the idea that changes in liver sensitivity to GH may underlie the decline in plasma Igf1 during food deprivation. In skeletal muscle, fasting downregulated gene transcripts encoding igf1, igfbp2b, igfbp5b, and igfbp6b, while also upregulating mRNAs for igf2 and ghr2. These data demonstrate isoform-specific regulation of Igfbps in liver and skeletal muscle in cabezon experiencing food deprivation and reinforce the idea that the repertoire of duplicated Igfbp genes that evolved in actinopterygian fishes supports a diverse scope of endocrine and paracrine functions.

Autophagy regulates the therapeutic potential of adipose-derived stem cells in LPS-induced pulmonary microvascular barrier damage

Adipose-derived stem cells (ADSCs) have been shown to be beneficial in some pulmonary diseases, and the paracrine effect is the major mechanism underlying ADSC-based therapy. Autophagy plays a crucial role in maintaining stem cell homeostasis and survival. However, the role of autophagy in mediating ADSC paracrine effects has not been thoroughly elucidated. We examined whether ADSCs participate in lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cell (PMVEC) barrier damage in a paracrine manner and illuminated the role of autophagy in regulating ADSC paracrine effects. PMVECs and ADSCs with or without autophagy inhibition were cocultured without intercellular contact, and the microvascular barrier function was assessed after LPS treatment. ADSC paracrine function was evaluated by detecting essential growth factors for endothelial cells. For in vivo experiments, ADSCs with or without autophagy inhibition were transplanted into LPS-induced lung-injury mice, and lung injury was assessed. ADSCs significantly alleviated LPS-induced microvascular barrier injury. In addition, ADSC paracrine levels of VEGF, FGF, and EGF were induced by LPS treatment, especially in the coculture condition. Inhibiting autophagy weakened the paracrine function and the protective effects of ADSCs on microvascular barrier injury. Moreover, ADSC transplantation alleviated LPS-induced lung injury, and inhibiting autophagy markedly weakened the therapeutic effect of ADSCs on lung injury. Together, these findings show that ADSC paracrine effects play a vital protective role in LPS-induced pulmonary microvascular barrier injury. Autophagy is a positive mediating factor in the paracrine process. These results are helpful for illuminating the role and mechanism of ADSC paracrine effects and developing effective therapies in acute lung injury.

Harnessing the Properties of Biomaterial to Enhance the Immunomodulation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have great therapeutic potential for tissue engineering and regenerative medicine due to their multipotency and paracrine functions. However, shortly after in vivo implantation, MSCs tend to migrate to the lungs and undergo apoptosis, which impairs their clinical efficacy. In addition, the ex vivo two-dimensional expansion of MSCs results in changes in their immunophenotype and functional activities compared to those in vivo. The use of biomaterials to culture and deliver MSCs has the potential to overcome these limitations. MSC-biomaterial constructs retain MSCs in situ and prolong their survival, while the MSCs ameliorate the foreign body reaction and fibrosis caused by the biomaterial. Biomaterial scaffolds can both preserve the tissue architecture and provide a three-dimensional biomimetic milieu for embedded MSCs, which enhance their paracrine functions, including their immunomodulatory potential. The dimensionality, physical characteristics, topographical cues, biochemistry, and microstructure can enhance the immunomodulatory potential of MSCs. Here, we review the link between the properties of biomaterial and the immunomodulatory potential of MSCs. Impact Statement Regeneration of cells, tissues, and whole organs is challenging. Mesenchymal stem cells (MSCs) have therapeutic potential in tissue engineering and regenerative medicine due to their paracrine functions, including immunomodulatory activity. The dimensionality, physical characteristics, topographical cues, biochemistry, and microstructure of biomaterial can be harnessed to enhance the immunomodulatory potential of MSCs for tissue engineering, which will increase their clinical efficacy, particularly for immune-related diseases.

Depletion of Bone Marrow-Derived Fibrocytes Attenuates TAA-Induced Liver Fibrosis in Mice.

Bone marrow-derived fibrocytes (FC) represent a unique cell type, sharing features of both mesenchymal and hematopoietic cells. FC were shown to specifically infiltrate the injured liver and participate in fibrogenesis. Moreover, FC exert a variety of paracrine functions, thus possibly influencing the disease progression. However, the overall contribution of FC to liver fibrosis remains unclear. We aimed to study the effect of a specific FC depletion, utilizing a herpes simplex virus thymidine kinase (HSV-TK)/Valganciclovir suicide gene strategy. Fibrosis was induced by oral thioacetamide (TAA) administration in C57BL/6J mice. Hepatic hydroxyproline content was assessed for the primary readout. The HSV-TK model enabled the specific depletion of fibrocytes. Hepatic hydroxyproline content was significantly reduced as a result of the fibrocyte ablation (−7.8%; 95% CI: 0.7–14.8%; p = 0.033), denoting a reduced deposition of fibrillar collagens. Lower serum alanine transaminase levels (−20.9%; 95% CI: 0.4–36.9%; p = 0.049) indicate a mitigation of liver-specific cellular damage. A detailed mode of action, however, remains yet to be identified. The present study demonstrates a relevant functional contribution of fibrocytes to chronic toxic liver fibrosis, contradicting recent reports. Our results emphasize the need to thoroughly study the biology of fibrocytes in order to understand their importance for hepatic fibrogenesis.

P4143IL-6 production in the vascular adventitia and adventitia-media-crosstalk in neointima formation

Abstract Background The aim of this study was to analyze the impact of the adventitial layer on vascular remodeling processes and to define the underlying cellular mechanisms. Methods and results Morphometric analysis of human coronary arteries and of murine femoral arteries at several times following vascular intervention revealed a significant correlation of neointimal and adventitial thickening (R2=0.6845, P&lt;0.001 for human samples; R2=0.6845, P&lt;0.001 for human samples. Immunohistochemical staining for the proliferation marker Ki-67 was performed 7, 14, and 21 days following injury of the murine femoral artery. Formation of a neointimal lesion at 21 days was preceded by high adventitial proliferation rates at 7 and 14 days (85.00±6.041 Ki67+adventitial cells vs. 5.118±0.633 Ki-67+neointimal cells at 7d, P&lt;0.0014; 28.80±5.240 Ki-67+adv. cells vs. 19.40±2.468 Ki-67+neoint. cells at 14d, P&lt;0.006, n=17). Complete removal of the adventitial layer prevented neointima formation, attributing pivotal importance to the adventitial layer (luminal stenosis: 71.73±3.77% vs. 7.44±1.71%, n=5, P&lt;0.0001). Re-transplantation of the aortic adventitia of ubiquitously GFP expressing C57BL/6-Tg (CAG-EGFP)1Osb/Jmice around the medial vascular layer of the femoral artery where the native adventitia has been removed completely restored neointima formation. Importantly, only very view GFP+cells were present in the neointimal layer, indicating that a direct contribution of adventitial cells to the neointimal lesion represents an extremely rare event. To investigate a potential paracrine effect of the activated adventitial layer, we explanted adventitial transplants 14 days following injury and transplantation and incubated the respective samples in serum-free media for 24 hours. BrdU incorporation assays and scratch wound assays revealed significantly increased proliferation and migration rates of human coronary artery SMCs in response to the supernatant of adventitial transplants compared to the supernatant of control samples. Further secretome analyses of the same adventitial supernatants identified predominantly interleukin (IL)-6 to trigger SMC proliferation and migration. Accordingly, serum-free media incubated with adventitial grafts of IL-6−/− mice prevented SMC proliferation and migration. Transplantation of the adventitia of IL-6−/− mice into C57BL/6J wild type mice was not sufficient to trigger neointima formation. Plain old balloon angioplasty, bare metal stent implantation, or drug-eluting stent implantation in swine coronary arteries and analysis for Ki-67+ cell counts supported the hypothesis in the large animal model and a more clinical setting. Conclusion Acute vascular injury is followed by an expansion of cytokine-producing adventitial cells, whose paracrine function and especially whose release of IL-6 is essential for the subsequent induction of the proliferation and migration of local SMC and thus for neointima formation.

Abstract P2064: Changes in Inflammatory Markers Monitored Across Time After Bariatric Surgery in Severely Obese Patients

Adipose tissue is a higly active endocrine tissue and produce adipokines, peptides produced by adipose tissue and exerting autocrine, paracrine and endocrine function. Objective: to analyze changes (Δ) in serum levels of several adipokines and inflammatory markers in morbidly obese patients (MO) after bariatric surgery (BS) at several follow-up time points. Methods: Fifty-two MO (81% women, 40% hypertensives, age 43.9±8.9 yr, BMI 42.2±5.4 Kg/m 2 , waist circumference 131.6±12.1 cm) were evaluated prospectively before and 1,3,6 and 12 months (m) after BS, with assessment of anthropometric data and serum concentrations of several adipokines and inflammatory markers. BS technique: laparoscopic gastric by-pass (28 of 52) and sleeve gastrectomy (24 of 52). Results: at 12m body weight (-35.7±10.2 Kg) and waist circumference (-27.2±9.4 cm) decreased (p&lt;0.001). Two inflammatory markers, leptin and hsCRP, decreased at 1,3,6 and 12m (p&lt;0,005 for all). Adiponectin and angiopoetin-2 increased from month 3 on. (Table). These Δ were statistically significant when examined by an ANOVA with repeated measures, excepte for adiponectin (p=0.065). After adjusting for age, sex, and BS technique, the BS technique was statistically significant only for ΔhsCRP (p=0.035). Pearson correlations were explored. There were no statistically significant correlation between Δ body weight or Δ waist circumference with changes of any of these markers at any follow-up time point. Conclusions: there is a decrease in inflammatory markers 1 m after BS in MO patients with progressive decrease throughout 12 m. These changes are independent of the BS technique and of Δ in body weight and waist circumference.

Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

Tyrosol Facilitates Neovascularization by Enhancing Skeletal Muscle Cells Viability and Paracrine Function in Diabetic Hindlimb Ischemia Mice.

As one of the most severe manifestations of diabetes, vascular complications are the main causes of diabetes-related morbidity and mortality. Hyperglycemia induces systemic abnormalities, including impaired angiogenesis, causing diabetic patients to be highly susceptible in suffering hindlimb ischemia (HLI). Despite its severe prognosis, there is currently no effective treatment for diabetic HLI. Skeletal muscle cells secrete multiple angiogenic factors, hence, recently are reported to be critical for angiogenesis; however, hyperglycemia disrupted the paracrine function in skeletal muscle cells, leading to the impaired angiogenesis potential observed in diabetic patients. The present study showed that tyrosol, a phenylethanoid compound, suppresses accumulation of intracellular reactive oxygen species (ROS) caused by hyperglycemia, most plausibly by promoting heme oxygenase-1 (HO-1) expression in skeletal muscle cells. Consequently, tyrosol exerts cytoprotective function against hyperglycemia-induced oxidative stress in skeletal muscle cells, increases their proliferation vigorously, and simultaneously suppresses apoptosis. Furthermore, tyrosol grossly increases the secretion of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) from skeletal muscle cells. This leads to enhanced proliferation and migration capabilities of vascular endothelial and smooth muscle cells, two types of cells that are responsible in forming blood vessels, through cell-cell communication. Finally, in vivo experiment using the diabetic HLI mouse model showed that tyrosol injection into the gastrocnemius muscle of the ischemic hindlimb significantly enhances the formation of functional blood vessels and subsequently leads to significant recovery of blood perfusion. Overall, our findings highlight the potential of the pharmacological application of tyrosol as a small molecule drug for therapeutic angiogenesis in diabetic HLI patients.

Canagliflozin Impairs Blood Reperfusion of the Ischemic Lower Limb by Inhibiting the Retention and Paracrine Function of Mesenchymal Stem Cells

Background: Canagliflozin (CANA) administration increases amputation risk of the lower limb in the clinic. The present study aimed to investigate whether and how CANA interferer with intracellular physiological processes of mesenchymal stem cells (MSCs), and its contribution to ischemic lower limb. Methods: The in vivo blood flow recovery of ischemic lower limb following CANA treatment was evaluated. The cellular function of MSCs after CANA treatment were also assessed in vitro. In silico docking analysis and mutant substitution assay were conducted to confirm the interaction of CANA and glutamate dehydrogenase 1 (GDH1). Findings: Following CANA treatment, attenuated angiogenesis and hampered recovery of blood flow were observed in ischemic region, and the proliferation inhibition, migration inhibition, and apoptosis of MSCs was evidenced. CANA was involved in MSC mitochondrial respiratory malfunction, ATP production inhibition, cytochrome c release and vessel endothelial growth factor A (VEGFA) secretion inhibition, which contribute to reduced tissue repair capacity of MSCs, resulting in compromised angiogenesis and blood reperfusion in ischemic region. The detrimental effects of CANA on MSCs result from the inhibition of GDH1 by CANA (evidenced by in silico docking analysis and H199A-GDH1/ N392A-GDH1 mutant substitution). Interpretation: Our work highlights that GDH1 activity inhibition by CANA interferes with metabolic activity of mitochondria, which deteriorates retention and VEGFA secretion of MSCs, hampering angiogenesis and the recovery of blood flow following lower limb ischemia. Funding Statement: This study was financially supported by National Natural Science Foundation of China (grant # 81800314 for J. N.), Natural Science Foundation of Zhejiang Province (grant # LQ18H090007 for Z. L.) and Wenzhou Science and Technology Bureau Foundation (grant # Y20190163 for Y. L.). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All experimental protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University.

Abstract 415: Electrical Stimulation of Pediatric Cardiac-derived c-kit + Progenitor Cells Improves Retention and Cardiac Function in Right Ventricular Heart Failure

Nearly 1% of babies born in the US will be diagnosed with a congenital heart defect (CHD). While surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. While primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Cardiac-derived c-kit + progenitor cells (CPCs) have been widely studied as a cell-based therapy for cardiac pathologies; however, unless these cells are extracted at a very young age their therapeutic efficacy has been shown to be diminished. Finding novel ways to enhance the reparative potential of these cells is thus of critical significance. We previously reported pediatric CPCs (isolated from patients between 1-5 years of age) respond to electrical stimulation (ES) by initiating intracellular calcium oscillations. Here, we investigate the ability of ES to enhance the retention and therapeutic function of pediatric CPCs in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES-CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes, such as integrins β1 and β5, and significantly increased in vitro survival and adhesion of cells. Lastly, we show that ES induces CPCs to release higher levels of reported pro-reparative factors in vitro, including angiogenin, FGF, HGF, and VEGF. These findings suggest electrical stimulation can be utilized to increase the retention, survival, and therapeutic effect of human c- kit + progenitor cells. Furthermore, our insights into the mechanisms of increased retention and paracrine function of ES-CPCs can have implications on a variety of cell-based therapies.

Mesenchymal Stem Cell Secretome: Cell-free Therapeutic Strategy in Regenerative Medicine

BACKGROUND: Mesenchymal stem (stromal) cells (MSCs) have a multipotent character, able to differentiate into several cell types, thus MSC serve as a cell reservoir for regenerative medicine. MSC therapeutic potency more associated to their immunosuppressive and anti-inflammatory properties rather than the multipotency, by its mechanism to secrete soluble factors with paracrine actions.CONTENT: MSC paracrine function was known to mediated partly by extracellular vesicles (EVs), which were released predominantly from the endosomal compartment contained in MSC secretome. EV contain a cargo bring micro RNA (miRNA), messenger RNA (mRNA), and proteins from their cells of origin, propose EV as a novel alternative to whole cell therapies, regarding the benefit of EV in safety and easier storage compared to the parent cells.SUMMARY: The discovery of EVs including exosomes in MSC secretome as key of stem cells beneficial function lead to the future hope of using cell-free regenerative therapies.KEYWORDS: MSC, secretome, conditioned media, extracellular vesicle, exosome

Mesenchymal Stem Cell Migration and Tissue Repair.

Mesenchymal stem cells (MSCs) are multilineage cells with the ability to self-renew and differentiate into a variety of cell types, which play key roles in tissue healing and regenerative medicine. Bone marrow-derived mesenchymal stem cells (BMSCs) are the most frequently used stem cells in cell therapy and tissue engineering. However, it is prerequisite for BMSCs to mobilize from bone marrow and migrate into injured tissues during the healing process, through peripheral circulation. The migration of BMSCs is regulated by mechanical and chemical factors in this trafficking process. In this paper, we review the effects of several main regulatory factors on BMSC migration and its underlying mechanism; discuss two critical roles of BMSCs—namely, directed differentiation and the paracrine function—in tissue repair; and provide insight into the relationship between BMSC migration and tissue repair, which may provide a better guide for clinical applications in tissue repair through the efficient regulation of BMSC migration.