The antidiabetic effect of mesenchymal stem cells is unrelated to their transdifferentiation potential but to their capability to restore Th1/Th2 balance and to modify the pancreatic microenvironment.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic ß-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.

Renal peritumoral adipose tissue undergoes a browning process and stimulates the expression of epithelial-mesenchymal transition markers in human renal cells.

Tumor cells can interact with neighboring adipose cells and adipocyte dedifferentiation appears to be an important aspect of tumorigenesis. We evaluated the size of adipocytes in human adipose explants from normal (hRAN) and kidney cancer (hRAT); changes in the expression of WAT and BAT/beige markers in hRAN and hRAT; the expression of epithelial-mesenchymal transition (EMT) cell markers in human kidney tumor (786-O, ACHN and Caki-1); and non-tumor (HK-2) epithelial cell lines incubated with the conditioned media (CMs) of hRAN and hRAT. We observed that hRAT adipocytes showed a significantly minor size compared to hRAN adipocytes. Also, we observed that both Prdm16 and Tbx1 mRNA and the expression of UCP1, TBX1, PPARγ, PCG1α, c/EBPα LAP and c/EBPα LIP was significantly higher in hRAT than hRAN. Finally, we found an increase in vimentin and N-cadherin expression in HK-2 cells incubated for 24 h with hRAT-CMs compared to hRAN- and control-CMs. Furthermore, desmin and N-cadherin expression also increased significantly in 786-O when these cells were incubated with hRAT-CMs compared to the value observed with hRAN- and control-CMs. We observed a significant decrease in E-cadherin expression in the ACHN cell line incubated with hRAT-CMs versus hRAN- and control-CMs. However, we did not observe changes in E-cadherin expression in HK-2, 786-O or Caki-1. The results obtained, together with the results previously published by our group, allow us to conclude that perirenal white adipose tissue browning contributes to tumor development in kidney cancer. In addition, hRAT-CMs increases the expression of mesenchymal markers in renal epithelial cells, which could indicate a regulation of EMT due to this adipose tissue.

Human adipose-derived mesenchymal stem cell-conditioned medium ameliorates polyneuropathy and foot ulceration in diabetic BKS db/db mice.

BACKGROUND: Diabetic polyneuropathy (DPN) is the most common and early developing complication of diabetes mellitus, and the key contributor for foot ulcers development, with no specific therapies available. Different studies have shown that mesenchymal stem cell (MSC) administration is able to ameliorate DPN; however, limited cell survival and safety reasons hinder its transfer from bench to bedside. MSCs secrete a broad range of antioxidant, neuroprotective, angiogenic, and immunomodulatory factors (known as conditioned medium), which are all decreased in the peripheral nerves of diabetic patients. Furthermore, the abundance of these factors can be boosted in vitro by incubating MSCs with a preconditioning stimulus, enhancing their therapeutic efficacy. We hypothesize that systemic administration of conditioned medium derived from preconditioned MSCs could reverse DPN and prevent foot ulcer formation in a mouse model of type II diabetes mellitus. METHODS: Diabetic BKS db/db mice were treated with systemic administration of conditioned medium derived from preconditioned human MSCs; conditioned medium derived from non-preconditioned MSCs or vehicle after behavioral signs of DPN was already present. Conditioned medium or vehicle administration was repeated every 2 weeks for a total of four administrations, and several functional and structural parameters characteristic of DPN were evaluated. Finally, a wound was made in the dorsal surface of both feet, and the kinetics of wound closure, re-epithelialization, angiogenesis, and cell proliferation were evaluated. RESULTS: Our molecular, electrophysiological, and histological analysis demonstrated that the administration of conditioned medium derived from non-preconditioned MSCs or from preconditioned MSCs to diabetic BKS db/db mice strongly reverts the established DPN, improving thermal and mechanical sensitivity, restoring intraepidermal nerve fiber density, reducing neuron and Schwann cell apoptosis, improving angiogenesis, and reducing chronic inflammation of peripheral nerves. Furthermore, DPN reversion induced by conditioned medium administration enhances the wound healing process by accelerating wound closure, improving the re-epithelialization of the injured skin and increasing blood vessels in the wound bed in a skin injury model that mimics a foot ulcer. CONCLUSIONS: Studies conducted indicate that MSC-conditioned medium administration could be a novel cell-free therapeutic approach to reverse the initial stages of DPN, avoiding the risk of lower limb amputation triggered by foot ulcer formation and accelerating the wound healing process in case it occurs.

Human renal adipose tissue from normal and tumor kidney: its influence on renal cell carcinoma.

Tumor cells can interact with neighboring adipose tissue. We evaluated components present in human adipose explants from normal (hRAN) and kidney cancer (hRAT) tissue, and we evaluated the effects of conditioned media (CMs) from hRAN and hRAT on proliferation, adhesion and migration of tumor and non-tumor human renal epithelial cell lines. In addition, we evaluated the expression of AdipoR1, ObR, CD44, vimentin, pERK and pPI3K on cell lines incubated with CMs. hRAN were obtained from healthy operated donors, and hRAT from patients who underwent a nephrectomy. hRAT showed increased levels of versican, leptin and ObR; and decreased levels of perilipin, adiponectin and AdipoR1, compared to hRAN. Cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRAT-CMs vs. hRAN- or control-CMs. Surprisingly, HK-2, 786-O and ACHN cells showed a significant decrease in cell migration after incubation with hRAN-CMs vs. control-CMs. No difference in proliferation of cell lines was found after 24 or 48 h of treatment with CMs. AdipoR1 in ACHN and Caki-1 cells decreased significantly after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. ObR and CD44 increased in tumor line cells, and vimentin increased in non-tumor cells, after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. We observed an increase in the expression of pERK and pPI3K in HK-2, 786-O and ACHN, incubated with hRAT-CMs. In conclusion, results showed that adipose microenvironment can regulate the behavior of tumor and non tumor human renal epithelial cells.

Characterization of diabetic neuropathy progression in a mouse model of type 2 diabetes mellitus.

Diabetes mellitus (DM) is one of most common chronic diseases with an increasing incidence in most countries. Diabetic neuropathy (DN) is one of the earliest and main complications of diabetic patients, which is characterized by progressive, distal-to-proximal degeneration of peripheral nerves. The cellular and molecular mechanisms that trigger DN are highly complex, heterogeneous and not completely known. Animal models have constituted a valuable tool for understanding diabetes pathophysiology; however, the temporal course of DN progression in animal models of type 2 diabetes (T2DM) is not completely understood. In this work, we characterized the onset and progression of DN in BKS diabetic (db/db) mice, including the main functional and histological features observed in the human disease. We demonstrated that diabetic animals display progressive sensory loss and electrophysiological impairments in the early-to-mid phases of the disease. Furthermore, we detected an early decrease in intraepidermal nerve fiber (IENF) density in 18-week-old diabetic mice, which is highly associated with sensory loss and constitutes a reliable marker of DN. Other common histological parameters of DN - like Schwann cells apoptosis and infiltration of CD3+ cells in the sciatic nerve - were altered in mid-to-late phases of the disease. Our results support the general consensus that DN evolves from initial functional to late structural changes. This work aimed to characterize the progression of DN in a reliable animal model sharing the main human disease features, which is necessary to assess new therapies for this complex disease. Finally, we also aimed to identify an effective temporal window where these potential treatments could be successfully applied.

Circulating miR-19b and miR-181b are potential biomarkers for diabetic cardiomyopathy.

Diabetic cardiomyopathy is characterized by metabolic changes in the myocardium that promote a slow and silent dysfunction of muscle fibers, leading to myocardium remodelling and heart failure, independently of the presence of coronary artery diseases or hypertension. At present, no imaging methods allow an early diagnosis of this disease. Circulating miRNAs in plasma have been proposed as biomarkers in the prognosis of several cardiac diseases. This study aimed to determine whether circulating miRNAs could be potential biomarkers of diabetic cardiomyopathy. Mice that were fed with a high fat diet for 16 months, showed metabolic syndrome manifestations, cardiac hypertrophy (without hypertension) and a progressive cardiac function decline. At 16 months, when maximal degree of cardiac dysfunction was observed, 15 miRNAs from a miRNA microarray screening in myocardium were selected. Then, selected miRNAs expression in myocardium (at 4 and 16 months) and plasma (at 4, 12 and 16 months) were measured by RT-qPCR. Circulating miR-19b-3p and miR-181b-5p levels were associated with myocardium levels during the development of diabetic cardiomyopathy (in terms of cardiac dysfunction), suggesting that these miRNAs could be suitable biomarkers of this disease in asymptomatic diabetic patients.

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

Regenerative Potential of Mesenchymal Stromal Cells: Age-Related Changes.

Preclinical and clinical studies have shown that a therapeutic effect results from mesenchymal stromal cells (MSCs) transplant. No systematic information is currently available regarding whether donor age modifies MSC regenerative potential on cutaneous wound healing. Here, we evaluate whether donor age influences this potential. Two different doses of bone marrow MSCs (BM-MSCs) from young, adult, or old mouse donors or two doses of their acellular derivatives mesenchymal stromal cells (acd-MSCs) were intradermally injected around wounds in the midline of C57BL/6 mice. Every two days, wound healing was macroscopically assessed (wound closure) and microscopically assessed (reepithelialization, dermal-epidermal junction, skin appendage regeneration, granulation tissue, leukocyte infiltration, and density dermal collagen fibers) after 12 days from MSC transplant. Significant differences in the wound closure kinetic, quality, and healing of skin regenerated were observed in lesions which received BM-MSCs from different ages or their acd-MSCs compared to lesions which received vehicle. Nevertheless, our data shows that adult's BM-MSCs or their acd-MSCs were the most efficient for recovery of most parameters analyzed. Our data suggest that MSC efficacy was negatively affected by donor age, where the treatment with adult's BM-MSCs or their acd-MSCs in cutaneous wound promotes a better tissue repair/regeneration. This is due to their paracrine factors secretion.

Dexamethasone and rosiglitazone are sufficient and necessary for producing functional adipocytes from mesenchymal stem cells.

The final product of adipogenesis is a functional adipocyte. This mature cell acquires the necessary machinery for lipid metabolism, loses its proliferation potential, increases its insulin sensitivity, and secretes adipokines. Multipotent mesechymal stromal cells have been recognized as a source of adipocytes both in vivo and in vitro. The in vitro adipogenic differentiation of human MSC (hMSC) has been induced up to now by using a complex stimulus which includes dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin (a classical cocktail) and evaluated according to morphological changes. The present work was aimed at demonstrating that the simultaneous activation of dexamethasone's canonical signaling pathways, through the glucocorticoid receptor and CCAAT-enhancer-binding proteins (C/EBPs) and rosiglitazone through peroxisome proliferator-activated receptor gamma (PPAR-gamma) is sufficient yet necessary for inducing hMSC adipogenic differentiation. It was also ascertained that hMSC exposed just to dexamethasone and rosiglitazone (D&R) differentiated into cells which accumulated neutral lipid droplets, expressed C/EBP-alpha, PPAR-gamma, aP2, lipoprotein lipase, acyl-CoA synthetase, phosphoenolpyruvate carboxykinase, adiponectin, and leptin genes but did not proliferate. Glucose uptake was dose dependent on insulin stimulus and high levels of adipokines were secreted (i.e. displaying not only the morphology but also expressing mature adipocytes' specific genes and functional characteristics). This work has demonstrated that (i) the activating C/EBPs and PPAR-gamma signaling pathways were sufficient to induce adipogenic differentiation from hMSC, (ii) D&R producing functional adipocytes from hMSC, (iii) D&R induce adipogenic differentiation from mammalian MSC (including those which are refractory to classical adipogenic differentiation stimuli). D&R would thus seem to be a useful tool for MSC characterization, studying adipogenesis pathways and producing functional adipocytes.

P450 CYP2C epoxygenase and CYP4A omega-hydroxylase mediate ciprofibrate-induced PPARalpha-dependent peroxisomal proliferation.

Peroxisomal proliferators, such as ciprofibrate, are used extensively as effective hypolipidemic drugs. The effects of these compounds on lipid metabolism require ligand binding activation of the peroxisome proliferator-activated receptor (PPAR) alpha subtype of nuclear receptors and involve transcriptional activation of the metabolic pathways involved in lipid oxidative metabolism, transport, and disposition. omega-Hydroxylated-eicosatrienoic acids (HEETs), products of the sequential metabolism of arachidonic acid (AA) by the cytochrome P450 CYP2C epoxygenase and CYP4A omega-hydroxylase gene subfamilies, have been identified as potent and high-affinity ligands of PPARalpha in vitro and as PPARalpha activators in transient transfection assays. Using isolated rat hepatocytes in culture, we demonstrate that specific inhibition of either the CYP2C epoxygenase or the CYP4A omega-hydroxylase abrogates ciprofibrate-induced peroxisomal proliferation, whereas inhibition of other eicosanoid-synthesizing pathways had no effect. Conversely, overexpression of the rat liver CYP2C11 epoxygenase leads to spontaneous peroxisomal proliferation, an effect that is reversed by a CYP inhibitor. Based on these results, we propose that HEETs may serve as endogenous PPARalpha ligands and that the P450 AA monooxygenases participate in ciprofibrate-induced peroxisomal proliferation and the activation of PPARalpha downstream targets.

Peroxisome proliferator-activated receptor gamma is a novel target of the nerve growth factor signaling pathway in PC12 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is subject to considerable interest because of its role in adipocyte differentiation, metabolic control, and anti-inflammatory action. PPARgamma research in brain cells is presently focused on glial PPARgamma because of its potential as a pharmacological target in the treatment of neurodegenerative diseases with an inflammatory component. In neurons PPARgamma function is far from clear, and PPARgamma agonist-dependent and -independent effects on cell survival or differentiation have been reported. We used PC12 cells, widely used to study neuronal signaling, such as nerve growth factor (NGF)-induced differentiation and survival or epidermal growth factor-dependent cell proliferation to dissect the possible involvement of PPARgamma in these pathways. We show that NGF but not epidermal growth factor increases the transcriptional activity of PPARgamma, and modulates the expression of this transcription factor. Because NGF signals through the tyrosine kinase (TrkA) NGF receptor and/or the p75NTR receptor, we used rescue experiments with a PC12 cell mutant lacking TrkA to show that NGF-induced PPARgamma activation is dependent on TrkA activation. Our results point out PPARgamma as a novel target of the TrkA-mediated neuronal cell survival and differentiating pathway and suggest a potential new inflammatory-independent therapeutic approach for pharmacological intervention in neurological disorders.