Adipose tissue macrophage populations and inflammation are associated with systemic inflammation and insulin resistance in obesity.

Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 nondiabetic adults across a range of body mass indexes (BMI 20.5-45.8 kg/m2). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (SI) and ß cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, SI, and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced SI, and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with SI, and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements.NEW AND NOTEWORTHY Adipose inflammation is proposed to be at the nexus of the systemic inflammation and metabolic derangements associated with obesity. The present study provides evidence to support adipose inflammation as a central feature of the pathophysiology of obesity. Adipose inflammation is associated with systemic and peripheral metabolic derangements, including increased systemic inflammation, reduced insulin sensitivity, and reduced skeletal muscle mitochondrial respiration.

HDL Cholesterol Efflux Does Not Predict Cardiovascular Risk in Hemodialysis Patients.

The cardioprotective effect of HDL is thought to be largely determined by its cholesterol efflux capacity, which was shown to inversely correlate with atherosclerotic cardiovascular disease in populations with normal kidney function. Patients with ESRD suffer an exceptionally high cardiovascular risk not fully explained by traditional risk factors. Here, in a post hoc analysis in 1147 patients with type 2 diabetes mellitus on hemodialysis who participated in the German Diabetes Dialysis Study (4D Study), we investigated whether the HDL cholesterol efflux capacity is predictive for cardiovascular risk. Efflux capacity was quantified by incubating human macrophage foam cells with apoB-depleted serum. During a median follow-up of 4.1 years, 423 patients reached the combined primary end point (composite of cardiac death, nonfatal myocardial infarction, and stroke), 410 patients experienced cardiac events, and 561 patients died. Notably, in Cox regression analyses, we found no association of efflux capacity with the combined primary end point (hazard ratio [HR], 0.96; 95% confidence interval [95% CI], 0.88 to 1.06; P=0.42), cardiac events (HR, 0.92; 95% CI, 0.83 to 1.02; P=0.11), or all-cause mortality (HR, 0.96; 95% CI, 0.88 to 1.05; P=0.39). In conclusion, HDL cholesterol efflux capacity is not a prognostic cardiovascular risk marker in this cohort of patients with diabetes on hemodialysis.

The anti-inflammatory function of HDL is impaired in type 2 diabetes: role of hyperglycemia, paraoxonase-1 and low grade inflammation.

BACKGROUND: Functional properties of high density lipoproteins (HDL) are increasingly recognized to play a physiological role in atheroprotection. Type 2 diabetes mellitus (T2DM) is characterized by low HDL cholesterol, but the effect of chronic hyperglycemia on the anti-inflammatory capacity of HDL, a metric of HDL function, is unclear. Therefore, the aim of the present study was to establish the impact of T2DM on the HDL anti-inflammatory capacity, taking paraoxonase-1 (PON-1) activity and low grade inflammation into account. METHODS: The HDL anti-inflammatory capacity, determined as the ability to suppress tumor necrosis factor-α (TNF-α) induced vascular cell adhesion molecule-1 (VCAM-1) mRNA expression in endothelial cells in vitro (higher values indicate lower anti-inflammatory capacity), PON-1 (arylesterase) activity, hs-C-reactive protein (hs-CRP), serum amyloid A (SAA) and TNF-α were compared in 40 subjects with T2DM (no insulin or statin treatment) and 36 non-diabetic subjects. RESULTS: T2DM was associated with impaired HDL anti-inflammatory capacity (3.18 vs 1.05 fold increase in VCAM-1 mRNA expression; P < 0.001), coinciding with decreased HDL cholesterol (P = 0.001), apolipoprotein A-I (P = 0.038) and PON-1 activity (P = 0.023), as well as increased hs-CRP (P = 0.043) and TNF-α (P = 0.005). In all subjects combined, age- and sex-adjusted multivariable linear regression analysis demonstrated that impaired HDL anti-inflammatory capacity was associated with hyperglycemia (ß = 0.499, P < 0.001), lower PON-1 activity (ß = - 0.192, P = 0.030) and higher hs-CRP (ß = 0.220, P = 0.016). CONCLUSIONS: The HDL anti-inflammatory capacity is substantially impaired in T2DM, at least partly attributable to the degree of hyperglycemia, decreased PON-1 activity and enhanced low grade chronic inflammation. Decreased anti-inflammatory protection capacity of HDL conceivably contributes to the increased atherosclerosis risk associated with T2DM.

The fractionation of adipose tissue procedure to obtain stromal vascular fractions for regenerative purposes.

Autologous adipose tissue transplantation is clinically used to reduce dermal scarring and to restore volume loss. The therapeutic benefit on tissue damage more likely depends on the stromal vascular fraction of adipose tissue than on the adipocyte fraction. This stromal vascular fraction can be obtained by dissociation of adipose tissue, either enzymatically or mechanical. Enzymatic dissociation procedures are time-consuming and expensive. Therefore, we developed a new inexpensive mechanical dissociation procedure to obtain the stromal vascular fraction from adipose tissue in a time sparing way, which is directly available for therapeutic injection. This mechanical dissociation procedure is denoted as the fractionation of adipose tissue (FAT) procedure. The FAT procedure was performed in eleven patients. The composition of the FAT-stromal vascular fraction was characterized by immunohistochemistry. Adipose derived stromal cells isolated from the FAT-stromal vascular fraction were compared with adipose derived stromal cells isolated from nondissociated adipose tissue (control) for their CD-surface marker expression, differentiation and colony forming unit capacity. Case reports demonstrated the therapeutic effect of the FAT-stromal vascular fraction. The FAT-stromal vascular fraction is an enrichment of extracellular matrix containing a microvasculature and culturable adipose derived stromal cells. Adipose derived stromal cells isolated from FAT-stromal vascular fraction did not differ from adipose derived stromal cells isolated from the control group in CD-surface marker expression, differentiation and colony forming unit capacity. The FAT procedure is a rapid effective mechanical dissociation procedure to generate FAT-stromal vascular fraction ready for injection with all its therapeutic components of adipose tissue: it contains culturable adipose derived stromal cells embedded in their natural supportive extracellular matrix together with the microvasculature.

Acute responsiveness to single leg cycling in adults with obesity.

Obesity is associated with several skeletal muscle impairments which can be improved through an aerobic exercise prescription. The possibility that exercise responsiveness is diminished in people with obesity has been suggested but not well-studied. The purpose of this study was to investigate how obesity influences acute exercise responsiveness in skeletal muscle and circulating amino metabolites. Non-obese (NO; n = 19; 10F/9M; BMI = 25.1 ± 2.8 kg/m2 ) and Obese (O; n = 21; 14F/7M; BMI = 37.3 ± 4.6 kg/m2 ) adults performed 30 min of single-leg cycling at 70% of VO2 peak. 13 C6 -Phenylalanine was administered intravenously for muscle protein synthesis measurements. Serial muscle biopsies (vastus lateralis) were collected before exercise and 3.5- and 6.5-h post-exercise to measure protein synthesis and gene expression. Targeted plasma metabolomics was used to quantitate amino metabolites before and 30 and 90 min after exercise. The exercise-induced fold change in mixed muscle protein synthesis trended (p = 0.058) higher in NO (1.28 ± 0.54-fold) compared to O (0.95 ± 0.42-fold) and was inversely related to BMI (R2  = 0.140, p = 0.027). RNA sequencing revealed 331 and 280 genes that were differentially expressed after exercise in NO and O, respectively. Gene set enrichment analysis showed O had six blunted pathways related to metabolism, cell to cell communication, and protein turnover after exercise. The circulating amine response further highlighted dysregulations related to protein synthesis and metabolism in adults with obesity at the basal state and in response to the exercise bout. Collectively, these data highlight several unique pathways in individuals with obesity that resulted in a modestly blunted exercise response.

Basement membrane proteins modulate cell migration on bovine pericardium extracellular matrix scaffold.

Native bovine pericardium (BP) exhibits anisotropy of its surface ECM niches, with the serous surface (i.e., parietal pericardium) containing basement membrane components (e.g., Laminin, Col IV) and the fibrous surface (i.e., mediastinal side) being composed primarily of type I collagen (Col I). Native BP surface ECM niche anisotropy is preserved in antigen removed BP (AR-BP) extracellular matrix (ECM) scaffolds. By exploiting sideness (serous or fibrous surface) of AR-BP scaffolds, this study aims to determine the mechanism by which ECM niche influences human mesenchymal stem cells (hMSCs) migration. Human mesenchymal stem cells (hMSC) seeding on serous surface promoted more rapid cell migration than fibrous surface seeding. Gene analysis revealed that expression of integrin α3 and α11 were increased in cells cultured on serous surface compared to those on the fibrous side. Monoclonal antibody blockade of α3ß1 (i.e., laminin binding) inhibited early (i.e. ≤ 6 h) hMSC migration following serous seeding, while having no effect on migration of cells on the fibrous side. Blockade of α3ß1 resulted in decreased expression of integrin α3 by cells on serous surface. Monoclonal antibody blockade of α11ß1 (i.e., Col IV binding) inhibited serous side migration at later time points (i.e., 6-24 h). These results confirmed the role of integrin α3ß1 binding to laminin in mediating early rapid hMSCs migration and α11ß1 binding to Col IV in mediating later hMSCs migration on the serous side of AR-BP, which has critical implications for rate of cellular monolayer formation and use of AR-BP as blood contacting material for clinical applications.

Senolytic agents lessen the severity of abdominal aortic aneurysm in aged mice.

Age is a major risk factor for abdominal aortic aneurysm (AAA), for which treatment options are limited to surgical intervention for large AAA and watchful waiting for small aneurysms. However, the factors that regulate the expansion of aneurysms are unclear. Development of new therapeutic strategies to prevent or treat small aneurysms awaits a more thorough understanding of the etiology of AAA formation and progression with aging. A variety of structural and functional changes have been reported in aging vasculature, but emerging evidence implicates senescent cells in the formation of AAA through their paracrine effects on vascular wall cell populations. Here we show that aging is associated with transcriptional changes in abdominal aortic tissue consistent with loss of smooth muscle cells, leukocyte adhesion, inflammation, and accumulation of senescent cells in the vascular wall and surrounding perivascular adipose tissue. Furthermore, aged mice demonstrated anatomical and histopathological features of AAA development in response to administration of angiotensin II over 28 days. Importantly, in our study we sought to determine if reducing senescent cells could lessen the severity of AAA in aged mice. We find that pretreatment of aged mice with oral senolytic agents (dasatinib + quercetin) reduced senescent cell abundance in the arterial walls and surrounding tissues and lessened the severity of AAA in response to angiotensin II administration. These data provide important preliminary evidence supporting a role of senescent cells in age-related AAA formation and progression and suggest that strategies to reduce senescent cell burden hold promise to lessen AAA severity.

The secretome of senescent preadipocytes influences the phenotype and function of cells of the vascular wall.

Senescent cells accumulate in numerous tissues in several chronic conditions such as aging, obesity, and diabetes. These cells are in a state of irreversible cell-cycle arrest and secrete inflammatory cytokines, chemokines and other immune modulators that have paracrine effects on nearby tissues. Adipose tissue, in particular, harbors senescent cells, which have been linked with numerous chronic conditions and age-related comorbidities. Here we performed a series of in vitro experiments to determine the influence of senescent preadipocytes on key cell types found in vessel walls, including vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages (MQs), and adipose-derived stromal/stem cells (ASCs). Primary human preadipocytes were irradiated to trigger a senescence-like phenotype. VSMCs, ECs, MQs, and ASCs were exposed to conditioned media collected from irradiated preadipocytes or control preadipocytes. Additional experiments were performed where VSMCs, ECs, MQs, and ASCs were co-cultured with irradiated or control preadipocytes. The secretome of irradiated cells induced an inflammatory phenotype, decreased cell viability, disrupted proliferation and migration, and impaired metabolic function of these cell types in vitro. These maladaptive changes in response to senescent cell exposure provide early evidence in support of a hypothesis that senescent preadipocytes trigger phenotypic and functional changes in key cellular components of blood vessels that may contribute to vascular disease.

Distinct Influence of Omega-3 Fatty Acids on the Plasma Metabolome of Healthy Older Adults.

Omega-3 polyunsaturated fatty acids (n3-PUFA) are well recognized for their potent triglyceride-lowering effects, but the potential influence of these bioactive lipids on other biological processes, particularly in the context of healthy aging, remains unknown. With the goal of gaining new insight into some less well-characterized biological effects of n3-PUFAs in healthy older adults, we performed metabolomics of fasting peripheral blood plasma collected from 12 young adults and 12 older adults before and after an open-label intervention of n3-PUFA (3.9 g/day, 2.7 g eicosapentaenoic [EPA], 1.2 g docosahexaenoic [DHA]). Proton nuclear magnetic resonance (1H-NMR) based lipoprotein subclass analysis revealed the expected reduction in total triglyceride (TG), but also demonstrated that n3-PUFA supplementation reduced very low-density lipoprotein (VLDL) particle number, modestly increased high-density lipoprotein (HDL) cholesterol, and shifted the composition of HDL subclasses. Further metabolite profiling by 1H-NMR and mass spectrometry revealed pronounced changes in phospholipids, cholesterol esters, diglycerides, and triglycerides following n3-PUFA supplementation. Furthermore, significant changes in hydroxyproline, kynurenine, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) following n3-PUFA supplementation provide further insight into some less well-recognized biological effects of n3-PUFA supplementation, including possible effects on protein metabolism, the kynurenine pathway, and glucose metabolism.

Impaired Hedgehog-Gli1 Pathway Activity Underlies the Vascular Phenotype of Polycystic Kidney Disease.

Polycystic kidney disease (PKD) has been linked to abnormal structure/function of ciliary proteins, leading to renal dysfunction. Recently, attention has been focused in the significant vascular abnormalities associated with PKD, but the mechanisms underlying this phenomenon remain elusive. Here, we seek to define the molecular events regulating the angiogenic imbalance observed in PKD. Using micro computed tomography (n=7) and protein expression analysis (n=5), we assessed the vascular density and the angiogenic profile of noncystic organs in a well-established PKD rat model (Polycystic Kidney-PCK rat). Heart and lungs of PCK rats have reduced vascular density and decreased expression of angiogenic factors compared with wild type. Similarly, PCK-vascular smooth muscle cells (VSMCs; n=4) exhibited lower levels of vascular markers. Then, using small interfering RNA (n=4), we determined the role of the ciliary protein fibrocystin in wild type-VSMCs, a critical component/regulator of vascular structure and function. Reduction of fibrocystin in wild type-VSMCs (n=4) led to an abnormal angiogenic potential similar to that observed in PCK-VSMCs. Furthermore, we investigated the involvement of the hedgehog signaling, a pathway closely linked to the primary cilium and associated with vascular development, in PKD. Mechanistically, we demonstrated that impairment of the hedgehog signaling mediates, in part, this abnormal angiogenic phenotype. Lastly, overexpression of Gli1 in PCK-VSMCs (n=4) restored the expression levels of proangiogenic molecules. Our data support a critical role of fibrocystin in the abnormal vascular phenotype of PKD and indicate that a dysregulation of hedgehog may be responsible, at least in part, for these vascular deficiencies.

Effect of cyclic deformation on xenogeneic heart valve biomaterials.

Glutaraldehyde-fixed bovine pericardium is currently the most popular biomaterial utilized in the creation of bioprosthetic heart valves. However, recent studies indicate that glutaraldehyde fixation results in calcification and structural valve deterioration, limiting the longevity of bioprosthetic heart valves. Additionally, glutaraldehyde fixation renders the tissue incompatible with constructive recipient cellular repopulation, remodeling and growth. Use of unfixed xenogeneic biomaterials devoid of antigenic burden has potential to overcome the limitations of current glutaraldehyde-fixed biomaterials. Heart valves undergo billion cycles of opening and closing throughout the patient's lifetime. Therefore, understanding the response of unfixed tissues to cyclic loading is crucial to these in a heart valve leaflet configuration. In this manuscript we quantify the effect of cyclic deformation on cycle dependent strain, structural, compositional and mechanical properties of fixed and unfixed tissues. Glutaraldehyde-fixed bovine pericardium underwent marked cyclic dependent strain, resulting from significant changes in structure, composition and mechanical function of the material. Conversely, unfixed bovine pericardium underwent minimal strain and maintained its structure, composition and mechanical integrity. This manuscript demonstrates that unfixed bovine pericardium can withstand cyclic deformations equivalent to 6 months of in vivo heart valve leaflet performance.

Attenuated activation of the unfolded protein response following exercise in skeletal muscle of older adults.

Sarcopenia is linked with impaired adaptive responses to exercise in aging skeletal muscle. The unfolded protein response (UPR) is an important intramyocellular molecular response pathway that is activated by exercise. The influence of age on skeletal muscle adaptive UPR in response to exercise, and the relationship to other key exercise-responsive regulatory pathways is not well-understood. We evaluated age-related changes in transcriptional markers of UPR activation following a single bout of resistance exercise in 12 young (27 ± 5yrs) and 12 older (75 ± 5yrs) healthy men and women. At baseline, there were modest differences in expression of UPR-related genes in young and older adults. Following exercise, transcriptional markers of UPR pathway activation were attenuated in older adults compared to young based on specific salient UPR-related genes and gene set enrichment analysis. The coordination of post-exercise transcriptional patterns between the UPR pathway, p53/p21 axis of autophagy, and satellite cell differentiation were less evident in older compared to young adults. In conclusion, transcriptomic analysis revealed an age-related decline in the adaptive UPR transcriptional response following a single bout of exercise that could contribute to impaired exercise responsiveness with age.

Graft-specific immune tolerance is determined by residual antigenicity of xenogeneic extracellular matrix scaffolds.

Antigenicity remains the primary barrier towards expanding the use of unfixed xenogeneic biomaterials in clinical applications. An unfixed xenogeneic biomaterial devoid of antigenicity, with maintained structural and mechanical integrity, has potential to overcome the limitations of current clinically utilized glutaraldehyde-fixed xenogeneic biomaterials, such as heart valve bioprostheses. Unfortunately, the threshold level of residual antigenicity necessary to overcome graft-specific immune responses in unfixed xenogeneic tissue has yet to be determined. Furthermore, little information is known regarding the extent to which in vitro disruption of native ECM properties, resulting from decellularization or antigen removal procedures, are tolerated following in vivo implantation. This manuscript demonstrates that humoral adaptive immune responses are more sensitive to residual xenogeneic biomaterial antigen content than are cell-mediated adaptive responses. Critically, the threshold for tolerable residual antigenicity is identified, with removal of ≥92% of lipophilic antigens required to reduce adaptive immune responses to levels equivalent to glutaraldehyde fixed tissue. Finally, the results demonstrated that the innate immune system tolerates minor changes in protein organization provided that molecular structure is maintained. Antigen removed xenogeneic biomaterials achieving these in vitro success criteria induce in vivo adaptive and innate tolerance, while modulating pro-regenerative constructive remodeling. STATEMENT OF SIGNIFICANCE: Removal of antigenic components from candidate xenogeneic biomaterials is the primary success criteria for development of extracellular matrix (ECM) scaffolds in tissue engineering applications. Currently, the threshold level of residual biomaterial antigenicity required to overcome recipient graft-specific adaptive immune responses is unknown. Additionally, the extent to which the innate immune response tolerates changes to the native ECM, resulting from the ECM scaffold production process, has yet to be determined. This manuscript not only establishes the threshold for tolerable residual antigenicity, but also demonstrates that deviations in protein organization are tolerated by the innate immune system, provided macromolecular structure remains intact. In doing so, we provide the foundation of an immunologically-acceptable unfixed xenogeneic biomaterial for use in clinical applications.

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging.

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived from Different Species.

Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose-derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction. Stem Cells Translational Medicine 2017;6:22-30.

Recombinant human collagen-based microspheres mitigate cardiac conduction slowing induced by adipose tissue-derived stromal cells.

BACKGROUND: Stem cell therapy to improve cardiac function after myocardial infarction is hampered by poor cell retention, while it may also increase the risk of arrhythmias by providing an arrhythmogenic substrate. We previously showed that porcine adipose tissue-derived-stromal cells (pASC) induce conduction slowing through paracrine actions, whereas rat ASC (rASC) and human ASC (hASC) induce conduction slowing by direct coupling. We postulate that biomaterial microspheres mitigate the conduction slowing influence of pASC by interacting with paracrine signaling. AIM: To investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM). METHOD: Unipolar extracellular electrograms, derived from microelectrode arrays (8x8 electrodes) containing NRVM, co-cultured with ASC or ASC loaded microspheres, were used to determine conduction velocity (CV) and conduction heterogeneity. Conditioned medium (Cme) of (co)cultures was used to assess paracrine mechanisms. RESULTS: Microspheres did not affect CV in control (NRVM) monolayers. In co-cultures of NRVM and rASC, hASC or pASC, CV was lower than in controls (14.4±1.0, 13.0±0.6 and 9.0± 1.0 vs. 19.5±0.5 cm/s respectively, p<0.001). Microspheres loaded with either rASC or hASC still induced conduction slowing compared to controls (13.5±0.4 and 12.6±0.5 cm/s respectively, p<0.001). However, pASC loaded microspheres increased CV of NRVM compared to pASC and NRMV co-cultures (16.3±1.3 cm/s, p< 0.001) and did not differ from controls (p = NS). Cme of pASC reduced CV in control monolayers of NRVM (10.3±1.1 cm/s, p<0.001), similar to Cme derived from pASC-loaded microspheres (11.1±1.7 cm/s, p = 1.0). The presence of microspheres in monolayers of NRVM abolished the CV slowing influence of Cme pASC (15.9±1.0 cm/s, p = NS vs. control). CONCLUSION: The application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through interference with a secondary autocrine myocardial factor.

Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering.

Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-ß1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-ß1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-ß1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers.

Development of recombinant collagen-peptide-based vehicles for delivery of adipose-derived stromal cells.

Stem cell therapy is a promising approach for repair, remodeling and even regenerate tissue of otherwise irreparable damage, such as after myocardial infarction (aMI). A severe limitation of cardiac stem cell therapy is the generally poor retention of administered cells in the target tissue. In tissue repair the main mode of action of adipose tissue-derived stem cells (ADSC) is the production of various growth factors, cytokines, anti-inflammatory and anti-apoptotic factors that together augment repair, remodeling, and regeneration. In this experiment, we used recombinant collagen peptide (RCP) with additional integrin-binding motives and different crosslinkers. Formulated as 50-100 µm microspheres with bound ADSC, we hypothesized that this would improve ADSC retention and function. Crosslinking was performed with chemical crosslinkers (EDC and HMDIC) at high and low concentrations or by thermal treatment (DHT). ADSC adhesion, proliferation, apoptosis/necrosis, and gene expressions in two-dimensional and three-dimensional were analyzed. In addition, the effect of ADSC conditioned medium (ADSC-CM) on proapoptotic/sprouting HUVEC was examined. Our results show that all materials support cell adhesion in short time point, however, EDC-High crosslinker induced ADSC apoptosis/necrosis. Gene expression results revealed lower expression of proinflammatory genes in chemical crosslinked materials, despite EDC-High the proinflammatory genes expressions were similar or higher than TCPS. In addition, cultured ADSC on DHT crosslinked RCP showed a proinflammatory phenotype compared to TCPS. Sprouting assay results confirmed the protective effect of ADSC-CM derived from TCPS and HMDIC-High crosslinked RCP proapoptotic HUVEC. We conclude that ADSC adhere to the materials and maintain their therapeutic profile.

Therapeutic Prospect of Adipose-Derived Stromal Cells for the Treatment of Abdominal Aortic Aneurysm.

Aneurysm refers to the dilation of the vessel wall for more than 50%. Abdominal aortic aneurysm (AAA) refers to the dilation and weakening of all three layers of the abdominal aorta, which mostly occur infrarenally. The population aged above 50 years is at risk of AAA development, while a familiar history doubles the risk. Progression of AAA can cause immanent rupture of the vascular wall and has a high mortality and morbidity risk. They are additional risk factors for AAA development such as gender, smoking, and dyslipidemia. In general, pathological features of AAA include inflammation, degradation of the extracellular matrix (ECM), and smooth muscle cell apoptosis. The main pathophysiology of AAA development is still unknown. Besides available treatment modalities for large AAA, which associate with a high mortality risk, effective, alternative, and safer treatments are required, preferably already at an early stage of AAA. For the last decades, tissue engineering and regenerative medicine showed promising potential therapeutic effects for various (cardiovascular) diseases, including AAA. Adipose tissue-derived stromal cells (ADSC) are a candidate source of stem cells for regenerative medicine. ADSC are isolated from adipose tissue with low risk and are easily cultured and expanded while maintaining their multipotency. In addition, due to their differentiation capacity and trophic factor production, ADSC serve an important role in tissue engineering and regenerative medicine modalities. In this review, we will highlight the main pathobiology of AAA and introduce ADSC as a new promising therapeutic source for small AAA.

Are Doppler ultrasonography parameters symmetric between the right and left kidney?

BACKGROUND: Among numerous modalities applied for evaluation of kidney diseases, Doppler ultrasonography (DU) provides information about the hemodynamic status of the kidneys. Meanwhile, the variability in DU parameters of the right and left kidney is a matter of controversy. The aim of this study was to determine whether any difference exists between the DU indices of the right and left kidney. METHODS: Retrospectively, we collected DU findings of 25 healthy potential renal transplant donors. All donors underwent renal DU and multidetector computed tomographic angiography before donor nephrectomy. DU indices, including peak systolic volume (PSV), resistive index (RI), pulsatility index (PI), end-diastolic volume (EDV), and acceleration time (AT), were recorded. RESULTS: The median age of the donors was 27 (range 23-39) years. The median PSV, RI, EDV, and AT for the right kidney were 29 cm/sec, 0.59, 10.9 cm/sec, and 50 msec, respectively. For the left kidney, the median PSV, RI, EDV, and AT were, respectively, 26.8 cm/sec, 0.60, 10.6 cm/sec, and 43 msec. Among the DU indices, median PI of the right kidney was significantly different from that of the left kidney (1.02 versus 0.95, P = 0.01). CONCLUSION: In conclusion, the present study revealed that right kidney DU indices, except for PI, may not differ from those of the left kidney.