Background-The impact of diabetes mellitus on the cardiac regenerative potential of cardiac stem cells (CSCs) is unknown yet critical, given that individuals with diabetes mellitus may well require CSC therapy in the future. Using human and murine CSCs from diabetic cardiac tissue, we tested the hypothesis that hyperglycemic conditions impair CSC function. Methods and Results-CSCs cultured from the cardiac biopsies of patients with diabetes mellitus (hemoglobin A1c, 10±2%) demonstrated reduced overall cell numbers compared with nondiabetic sourced biopsies (P=0.04). When injected into the infarct border zone of immunodeficient mice 1 week after myocardial infarction, CSCs from patients with diabetes mellitus demonstrated reduced cardiac repair compared with nondiabetic patients. Conditioned medium from CSCs of patients with diabetes mellitus displayed a reduced ability to promote in vitro blood vessel formation (P=0.02). Similarly, conditioned medium from CSCs cultured from the cardiac biopsies of streptozotocin-induced diabetic mice displayed impaired angiogenic capacity (P=0.0008). Somatic gene transfer of the methylglyoxal detoxification enzyme, glyoxalase-1, restored the angiogenic capacity of diabetic CSCs (diabetic transgenic versus nondiabetic transgenic; P=0.8). Culture of nondiabetic murine cardiac biopsies under high (25 mmol/L) glucose conditions reduced CSC yield (P=0.003), impaired angiogenic (P=0.02) and chemotactic (P=0.003) response, and reduced CSC-mediated cardiac repair (P<0.05). Conclusions-Diabetes mellitus reduces the ability of CSCs to repair injured myocardium. Both diabetes mellitus andpreconditioning CSCs in high glucose attenuated the proangiogenic capacity of CSCs. In this study, we have investigated the effects of diabetes mellitus and a hyperglycemic environment on the function of ex vivo proliferated human and murine CSCs. Furthermore, we assessed the ability of GLO-1 overexpression to prevent and reverse hyperglycemia-induced CSC dysfunction. MethodsDetailed experimental methods are available in the online-only Data Supplement. CSC Isolation and CultureHuman CSCs were obtained from left atrial appendages donated by patients (aged 18-80 years) undergoing clinically indicated heart surgery after informed consent. Murine CSCs were obtained from cardiac tissue of wild-type C57Bl/6, C57Bl/6-cKit-EGFP, or C57Bl/6-PEP8-hGlo-1 transgenic mice (aged 2 to 12 months) under isoflurane sedation. CSCs were cultured as described previously. 19,20 Hyperglycemia was induced in C57BL/6 or PEP8-hGlo-1 mice by intraperitoneal injection of streptozotocin (50 mg/kg for 5 days) in 0.05 mol/L sodium citrate. Nondiabetic control mice received equal volumes of 0.05 mol/L sodium citrate. Fasting blood glucose measurements were obtained 10 to 14 days after the fifth streptozotocin injection and again before euthanasia. Mean fasting blood glucose at the time of euthanasia was 29.0±2.3 mmol/L for streptozotocin-injected animals versus 5.6±0.1 mmol/L for controls (P=0.0005). Diabetic Cardiac Explant GLO-1 and...

Adipose tissue can be regarded as a multidepot organ responsible for metabolic homeostasis by managing sophisticated energy transactions as well as by producing bioactive molecules that regulate insulin sensitivity and immune and vascular responses. Chronic nutrient excess expands adipose tissue, and concomitant variations in its cellular and matrix remodeling can affect the extent of the metabolic dysfunction that is associated with obesity. Preadipocytes, also termed adipose progenitor cells, play a pivotal role in determining whether a dysfunctional hypertrophic state arises as opposed to a hyperplastic process in which mature adipocytes remain relatively responsive. Obesity is associated with infiltration of macrophages, and these immune cells have been shown to communicate with preadipocytes to influence how they differentiate, survive, and proliferate. Understanding macrophage-preadipocyte interactions and their effect on adipose remodeling mechanisms may identify potential therapeutic molecular targets to improve adipose tissue function, even in the face of obesity.

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Molgat AS, Gagnon A, Sorisky A. Preadipocyte apoptosis is prevented by macrophage-conditioned medium in a PDGF-dependent manner. Am J Physiol Cell Physiol 296: C757-C765, 2009. First published February 18, 2009 doi:10.1152/ajpcell.00617.2008.-Obesity is associated with macrophage accumulation and inflammation in adipose tissue. Macrophage-secreted factors have been reported to inhibit the differentiation of preadipocytes into adipocytes and to modulate adipogenic extracellular matrix gene expression. To enlarge our understanding of macrophages and the scope of their interactions with preadipocytes, we investigated their effect on preadipocyte survival. Acute exposure of 3T3-L1 preadipocytes to J774A.1 macrophage-conditioned medium (MacCM) stimulated platelet-derived growth factor receptor (PDGFR) tyrosine phosphorylation by 4.1-fold. There were significant increases in the phosphocontent of downstream PDGFR targets Akt and ERK1/2 (5.3-fold and 2.4-fold, respectively) that were inhibited by PDGF immunoneutralization or by the selective PDGFR inhibitor imatinib. Serum-free J774A.1-MacCM or RAW264.7-MacCM completely prevented 3T3-L1 preadipocyte apoptosis normally induced by serum deprivation. Addition of PDGF alone to serum-free control medium was sufficient to prevent 3T3-L1 preadipocyte apoptosis. Inhibition of PDGFR activation by MacCM, either by addition of imatinib or by PDGF immunodepletion of MacCM, effectively disrupted the prosurvival effect. In summary, our data indicate that MacCM promotes preadipocyte survival in a PDGFdependent manner.platelet-derived growth factor; obesity; macrophage OBESITY, defined as an increase in adiposity, results from a chronic positive energy balance. Expansion of adipose tissue occurs through the formation of new adipocytes, via the differentiation of stromal precursor preadipocytes, and the enlargement of existing adipocytes (9). Deficiency in the number of preadipocytes, or their capacity to differentiate, may lead to excessively hypertrophied and dysfunctional adipocytes that overproduce immune cell chemoattractant proteins (7,13,14). Concomitantly, the population of adipose tissue macrophages rises due to monocyte infiltration. This abnormal cellular remodeling of adipose tissue is associated with low-grade inflammation and insulin resistance (38,39).Paracrine communication among macrophages, adipocytes, and preadipocytes is believed to contribute to obesity-associated adipose tissue dysfunction (12). We and others have reported that macrophages produce anti-adipogenic factors that restrain the differentiation of preadipocytes into adipocytes (4,5,15,17,33,40). Macrophage-secreted factors have also been observed to have variable effects on preadipocyte proliferation (17,23). Apoptosis, the other key preadipocyte fate pertinent to adipose tissue remodeling (11, 21, 28), has not been evaluated with respect to potential macrophage paracrine interactions.The precise identity of the macrophage products influencing preadipocyte responses is not known. Here, we demonstrate th...

Macrophage infiltration into adipose tissue, associated with obesity, is thought to contribute to abnormal adipose tissue remodeling, low-grade inflammation, and insulin resistance. Medium conditioned by macrophages (MacCM) inhibits 3T3-L1 and human adipocyte differentiation, as well as early adipogenic cell cycle events including MCE and retinoblastoma protein (Rb) phosphorylation. Our objective was to determine if the inhibition of Rb phosphorylation was linked to changes in cell cycle-related proteins. We treated 3T3-L1 preadipocytes with adipogenic inducers for 24 h in control medium versus J774A.1-MacCM. The differentiation-induced mRNA and protein expression of cyclin A, an activator of cyclin-dependent kinase (cdk) 2 which phosphorylates Rb, was inhibited by 82% and 73%, respectively, by J774A.1-MacCM; adipogenic expression of Myc, a transcriptional regulator of cyclin A, was also suppressed significantly. Consistent with the reduction in cyclin A levels, the activation of cdk2 by adipogenic inducers was inhibited by 75% by J774A.1-MacCM. J774A.1-MacCM also lowered levels of cyclins D1 and D2. Inhibition studies demonstrated that platelet-derived growth factor, an anti-adipogenic factor found in J774A.1-MacCM, was not responsible for the inhibitory effect on differentiation. The anti-adipogenic effect of J774A.1-MacCM was resistant to proteinase K and heat treatment, and was present in a <3 kDa fraction. Our data indicate that J774A.1-MacCM interferes with the upregulation of cyclin A levels and cdk2 activity that are required for Rb phosphorylation and MCE in 3T3-L1 adipogenesis.

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While cell therapy is emerging as a promising option for patients with ischemic cardiomyopathy (ICM), the influence of advanced donor age and a history of ischemic injury on the reparative performance of these cells are not well defined. As such, intrinsic changes that result from advanced donor age and ischemia are explored in hopes of identifying a molecular candidate capable of restoring the lost reparative potency of heart explant‐derived cells (EDCs) used in cell therapy. EDCs were cultured from myocardial biopsies obtained from young or old mice 4 weeks after randomization to experimental myocardial infarction or no intervention. Advanced donor age reduces cell yield while increasing cell senescence and the secretion of senescence‐associated cytokines. A history of ischemic injury magnifies these effects as cells are more senescent and have lower antioxidant reserves. Consistent with these effects, intramyocardial injection of EDCs from aged ischemic donors provided less cell‐mediated cardiac repair. A transcriptome comparison of ICM EDCs shows aging modifies many of the pathways responsible for effective cell cycle control and DNA damage/repair. Over‐expression of the barely explored antisenescent transcription factor, Mybl2, in EDCs from aged ICM donors reduces cell senescence while conferring salutary effects on antioxidant activity and paracrine production. In vivo, we observed an increase in cell retention and vasculogenesis after treatment with Mybl2‐over‐expressing EDCs which improved heart function in infarcted recipient hearts. In conclusion, Mybl2 over‐expression rejuvenates senescent EDCs sourced from aged ICM donors to confer cell‐mediated effects comparable to cells from young nonischemic donors.

Adipose tissue contains macrophages whose state of activation is regulated as obesity develops. Macrophagesecreted factors influence critical processes involved in adipose tissue homeostasis, including preadipocyte proliferation and differentiation into adipocytes. Macrophage-conditioned medium (MacCM) from J774A