Adoptive Transfer of Syngeneic Bone Marrow-Derived Cells in Mice with Obesity-Induced Diabetes
2009; Elsevier BV; Volume: 174; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080606
ISSN1525-2191
AutoresJun Chen, Houwei Li, Francesco Addabbo, Fung Zhang, Edward Pelger, Daniel Patschan, Hyeong Cheon Park, Mei‐Chuan Kuo, Jei Ni, Glenda C. Gobé, Praveen Chander, Alberto Nasjletti, Michael S. Goligorsky,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoThere are conflicting data regarding the effects of transplantation of bone marrow-derived cells (BMDCs) on the severity of diabetes. We therefore inquired whether the competence of BMDCs is preserved on adoptive transfer into diabetic (db/db) mice and how the adoptive transfer of BMDCs affects vascular and metabolic abnormalities in these mice. Recipient db/db mice received infusions of BMDCs prepared from either db/db or non-diabetic heterozygout mice (db/m) mice and effects on endothelium-dependent relaxation, insulin sensitivity, and renal function were evaluated. Recipients of BMDCs from db/m, but not db/db donors showed better glucose control, exhibited striking improvement in endothelium-dependent relaxation in response to acetylcholine, and had partially restored renal function. Improved glucose control was due to enhanced insulin sensitivity, most likely secondary to improved vascular function. Enhanced apoptosis of endothelial progenitor cells under oxidative stress, as well as decreased endothelial progenitor cell numbers were responsible for the apparent functional incompetence of BMDCs from db/db donors. Treatment of db/db mice with Ebselen restored the resistance of both BMDCs and endothelial progenitor cells to oxidative stress, improved acetylcholine-induced vasorelaxation, and reduced proteinuria in db/db recipients of BMDC transplantation. In conclusion, infusion of BMDCs obtained from db/m donors to db/db recipient mice benefited macrovascular function, insulin sensitivity, and nephropathy. BMDCs obtained from db/db mice were functionally incompetent secondary to the increased proportion of apoptotic cells on oxidative stress challenge; their competence was restored by Ebselen therapy. There are conflicting data regarding the effects of transplantation of bone marrow-derived cells (BMDCs) on the severity of diabetes. We therefore inquired whether the competence of BMDCs is preserved on adoptive transfer into diabetic (db/db) mice and how the adoptive transfer of BMDCs affects vascular and metabolic abnormalities in these mice. Recipient db/db mice received infusions of BMDCs prepared from either db/db or non-diabetic heterozygout mice (db/m) mice and effects on endothelium-dependent relaxation, insulin sensitivity, and renal function were evaluated. Recipients of BMDCs from db/m, but not db/db donors showed better glucose control, exhibited striking improvement in endothelium-dependent relaxation in response to acetylcholine, and had partially restored renal function. Improved glucose control was due to enhanced insulin sensitivity, most likely secondary to improved vascular function. Enhanced apoptosis of endothelial progenitor cells under oxidative stress, as well as decreased endothelial progenitor cell numbers were responsible for the apparent functional incompetence of BMDCs from db/db donors. Treatment of db/db mice with Ebselen restored the resistance of both BMDCs and endothelial progenitor cells to oxidative stress, improved acetylcholine-induced vasorelaxation, and reduced proteinuria in db/db recipients of BMDC transplantation. In conclusion, infusion of BMDCs obtained from db/m donors to db/db recipient mice benefited macrovascular function, insulin sensitivity, and nephropathy. BMDCs obtained from db/db mice were functionally incompetent secondary to the increased proportion of apoptotic cells on oxidative stress challenge; their competence was restored by Ebselen therapy. Transplantation of bone marrow-derived cells (BMDCs) has emerged as a promising tool in regenerative medicine. This heterogeneous cell population, consisting of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and endothelial progenitors, has the capacity to differentiate into cells of endothelial, epithelial, cardiomyocyte, and neuronal lineage.1Asahara T Masuda H Takahashi T Kalka C Pastore C Silver M Kearne M Magner M Isner JM Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ Res. 1999; 85: 221-228Crossref PubMed Scopus (2973) Google Scholar, 2Krause DS Theise ND Collector MI Henegariu O Hwang S Gardner R Neutzel S Sharkis SJ Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell.Cell. 2001; 105: 369-377Abstract Full Text Full Text PDF PubMed Scopus (2507) Google Scholar, 3Orlic D Kajstura J Chimenti S Jakoniuk I Anderson SM Li B Pickel J McKay R Nadal-Ginard B Bodine DM Leri A Anversa P Bone marrow cells regenerate infarcted myocardium.Nature. 2001; 410: 701-705Crossref PubMed Scopus (4746) Google Scholar, 4Brazelton TR Rossi FM Keshet GI Blau HM From marrow to brain: expression of neuronal phenotypes in adult mice.Science. 2000; 290: 1775-1779Crossref PubMed Scopus (1605) Google Scholar In a model of hindlimb ischemia, implantation of autologous BMDCs resulted in therapeutic angiogenesis and improved vascularization of the affected limb in both non-diabetic and diabetic rats.5Hirata K Li TS Nishida M Ito H Matsuzaki M Kasaoka S Hamano K Autologous bone marrow cell implantation as therapeutic angiogenesis for ischemic hindlimb in diabetic rat model.Am J Physiol Heart Circ Physiol. 2003; 284: H66-H70Crossref PubMed Scopus (61) Google Scholar In ApoE-deficient mice, transplantation of BMDCs resulted in the restoration of vascular functions.6Rauscher FM Goldschmidt-Clermont PJ Davis BH Wang T Gregg D Ramaswami P Pippen AM Annex BH Dong C Taylor DA Aging, progenitor cell exhaustion, and atherosclerosis.Circulation. 2003; 108: 457-463Crossref PubMed Scopus (654) Google Scholar There are conflicting data on the effect of transplantation of BMDCs on the severity of diabetes7Than S Ishida H Inaba M Fukuba Y Seino Y Adachi M Imura H Ikehara S Bone marrow transplantation as a strategy for treatment of non-insulin-dependent diabetes mellitus in KK-Ay mice.J Exp Med. 1992; 176: 1233-1238Crossref PubMed Scopus (68) Google Scholar, 8Ianus A Holz GG Theise ND Hussain MA In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.J Clin Invest. 2003; 111: 843-850Crossref PubMed Scopus (699) Google Scholar, 9Lechner A Yang YG Blacken RA Wang L Nolan AL Habener JF No evidence for significant transdifferentiation of bone marrow into pancreatic beta-cells in vivo.Diabetes. 2004; 53: 616-623Crossref PubMed Scopus (234) Google Scholar, 10Hasegawa Y Ogihara T Yamada T Ishigaki Y Imai J Uno K Gao J Kaneko K Ishihara H Sasano H Nakauchi H Oka Y Katagiri H Bone marrow (BM) transplantation promotes beta-cell regeneration after acute injury through BM cell mobilization.Endocrinology. 2007; 148: 2006-2015Crossref PubMed Scopus (99) Google Scholar and on the renal pathology and dysfunction in the murine model of renal fibrosis and ischemia-reperfusion injury.11Roufosse C Bou-Gharios G Prodromidi E Alexakis C Jeffery R Khan S Otto WR Alter J Poulsom R Cook HT Bone marrow-derived cells do not contribute significantly to collagen I synthesis in a murine model of renal fibrosis.J Am Soc Nephrol. 2006; 17: 775-782Crossref PubMed Scopus (87) Google Scholar, 12Broekema M Harmsen MC van Luyn MJ Koerts JA Petersen AH van Kooten TG van Goor H Navis G Popa ER Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats.J Am Soc Nephrol. 2007; 18: 165-175Crossref PubMed Scopus (154) Google Scholar Furthermore, recent data indicate that BMDC may become incompetent with regard to their ability to regenerate various tissues and organs.6Rauscher FM Goldschmidt-Clermont PJ Davis BH Wang T Gregg D Ramaswami P Pippen AM Annex BH Dong C Taylor DA Aging, progenitor cell exhaustion, and atherosclerosis.Circulation. 2003; 108: 457-463Crossref PubMed Scopus (654) Google Scholar, 13George J Afek A Abashidze A Shmilovich H Deutsch V Kopolovich J Miller H Keren G Transfer of endothelial progenitor and bone marrow cells influences atherosclerotic plaque size and composition in apolipoprotein E knockout mice.Arterioscler Thromb Vasc Biol. 2005; 25: 2636-2641Crossref PubMed Scopus (191) Google Scholar Taking into account multiple macro- and microvascular complications of diabetes, we inquired a) whether transplantation of BMDCs may affect some of these functional abnormalities, and b) whether the competence of BMDCs in diabetic mice is preserved. Here, we report a dramatic improvement of macrovascular dysfunction and insulin sensitivity in db/db mice recipients of syngeneic BMDC isolated from the non-diabetic mice (but not from their diabetic counterparts), and provide evidence for BMDC incompetence in diabetic animals. The animal study protocol was in accordance with National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services Public Health Services, NIH, NIH Publication No. 86–23, 1985) and approved by the Institutional Animal Care and Use Committee. The type II diabetic murine model db/db mice and db/m mice were obtained from Jackson Laboratory (Bar Harbor, Maine; C57BL/6 background). The body weight and blood glucose level of mice between ages of 8 to 16 weeks were monitored throughout the study. Briefly, bone marrow from male donor db/db and db/m mice was flushed under sterile conditions with Hank's balanced salt solution (HBSS) from the medullary cavities of tibiae and femurs using a 21-gauge needle. Whole bone marrow single cell suspension was fractionated using Histopaque-1077 solution (Sigma) gradient separation. Mononuclear cells were collected, washed, and checked for viability using trypan blue exclusion technique.8Ianus A Holz GG Theise ND Hussain MA In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.J Clin Invest. 2003; 111: 843-850Crossref PubMed Scopus (699) Google Scholar BMDCs were labeled with Cell Tracker (CM-DiI) (Invitrogen, Eugene, OR). In three independent experiments, male recipient db/db mice (age 16 weeks) received approximately 106 BMDC by tail vein injection. The db/db mice that received the BMDC from db/m mice are designated as dbTxm. The db/db mice that received the BMDC from db/db mice are designated as dbTxdb. The same transfusion procedure was repeated three times every 10 days. In additional series of experiments, db/db mice were treated with Ebselen by gavage, twice a day at 5 mg/kg/day, dissolved in 5% carboxymethyl (CM) cellulose suspension. This group of donor mice was labeled as dbEbs-in vivo and its corresponding recipient db/db mice were designated as TxdbEbs-in vivo. Control db/db mice received only CM cellulose (Sigma, St. Louis, MO) suspension (vehicle treatment; designated as dbCM and its corresponding BMDC recipient db/db mice designated as TxdbCM.) Another group of recipient db/db mice was transfused with BMDC of db/db origin, but treated with Ebs (1 μg/ml) overnight in full Dulbecco's Modified Eagle Medium (DMEM) medium at 37°C in CO2 incubator before transfusion. This group of recipient mice was designated as TxdbEbs-ex vivo. Because all animals had the same C57BL/6 background, no alloimmune or graft-versus-host response was expected (nor observed). Mice were euthanized 20 days after the final transfusion (at age 23 weeks) by intraperitoneal injection of ketamine/xylazine (60/7.7 mg/kg, respectively). A mid-laparotomy was performed and blood, thoracic aorta, kidney, and pancreas were harvested for further analyses. Thoracic aortas were cleared of periadventitial tissue and cut transversely into rings 1.5 to 2.0 mm in diameter. Vascular rings, handled carefully to avoid damage to the inner surface, were mounted on wires in the chambers of a multivessel myograph (J.P. Trading, Aarhus, Denmark) and bathed in Krebs' buffer. The medium was gassed with 95% O2 and 5% CO2 and maintained at 37°C (pH 7.4). After equilibration (30 minutes), the rings were set to an internal circumference equivalent to 90% of full relaxation under a transmural pressure of 100 mm Hg and allowed to stabilize for 20 to 30 minutes. The rings were then depolarized with potassium chloride (60 mmol/L) to evaluate maximal contraction. After washing with a Krebs' buffer, the vascular preparations were contracted with phenylephrine (10−6 mol/L), and when the contractile response was stabilized (steady-state phase, 12 to 15 minutes), vasorelaxing responses to cumulative increments in the concentration of acetylcholine or NONOate were examined.14Brodsky SV Gealekman O Chen J Zhang F Togashi N Crabtree M Gross SS Nasjletti A Goligorsky MS Prevention and reversal of premature endothelial cell senescence and vasculopathy in obesity-induced diabetes by ebselen.Circ Res. 2004; 94: 377-384Crossref PubMed Scopus (181) Google Scholar Plasma glucose was measured using glucometer (OneTouch Ultra, Lifescan) by collecting 2 μl blood through nicking the end of the tail. To estimate insulin resistance, we conducted insulin tolerance test and homeostasis model assessment (HOMA) index analysis, as previously described.15Francisco G Hernandez C Galard R Simo R Usefulness of homeostasis model assessment for identifying subjects at risk for hypoglycemia failure during the insulin hypoglycemia test.J Clin Endocrinol Metab. 2004; 89: 3408-3412Crossref PubMed Scopus (4) Google Scholar, 16Matthews DR Hosker JP Rudenski AS Naylor BA Treacher DF Turner RC Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (26555) Google Scholar Briefly, for the insulin tolerance test, animals were fasted for 3 hours. The mice were weighed and 1.5 units/kg body weight of diluted regular human insulin 1:1000 (0.1 inits/ml) was injected intraperitoneally. At 90 and 180 minutes, blood glucose was sampled. HOMA index was calculated by the formula: fasting plasma insulin × fasting plasma glucose/405. The Luminex multiplex assay (cat#: MCYTO-70K-PMX) was used for simultaneous quantification of the following mouse cytokines/chemokines in the plasma: interleukin (IL)-1α and β, IL-6, IL-9, IL-10, interferon (IFN)γ, interferone-gamma-inducible protein (IP-10), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNFβ), keratinocyte chemoattractant (KC), monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1β) and mRANTES. A mixture of beads was incubated with standards or plasma samples, followed by the appropriate biotinylated antibody and streptavidin-phycoerythrin reporter. Beads were analyzed using Luminex-100. Plasma insulin, amylin, glucagon, and leptin concentration were simultaneously measured using the same technique (cat#: MENDO-75K-05). Tissue samples of kidney and pancreas were fixed in a 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, followed by sequential incubation in 15% and 30% sucrose overnight at 4°C each. Embedding was performed in an optimal cutting temperature compound (Tissue-Tek, Torrance, CA), and embedded samples were stored at −80°C. Frozen samples were cut into 10-μm-thick sections (Cryomicrotom CM 1850, Leica Microsystems, Bannockburn, IL). Nonspecific protein binding was blocked by 1-hour incubation with PBS-bovine serum albumin (1%). The following primary antibodies were used: anti-mouse insulin (Santa Cruz Biotechnology, Santa Cruz, CA), CD31 (BD Pharmingen, San Jose, CA), and CD68 (Serotec, Oxford, UK). For CD-68 staining, horseradish peroxidase conjugated goat anti-rat IgG was used as the secondary antibody. Peroxidase activity was blocked by 15 minutes of incubation with peroxidase block solution (1:10; DakoCytomation, Glostrup, Denmark). To visualize the positive immunoreaction, the peroxidase substrate 3,3′-diaminobenzidine chromogen was used. Hematoxylin solution was used for counterstaining. Negative controls for all immunolabeling procedures were accomplished by incubation with 1% PBS-bovine serum albumin instead of the primary antibody. For immunofluorescence staining, fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used. Incubations with primary antibodies were performed overnight at 4°C and incubations with secondary antibodies were performed for 1 hour at room temperature. Control samples were stained with secondary antibodies only. To visualize the nuclei, tissue sections were counterstained with 4,6-diamidino-2-phenylindole (Molecular Probes). Sections were examined using a Nikon inverted fluorescence microscope (Eclipse TE2000-U) equipped with a digital camera (Spot model 4.2; Diagnostic Instruments, Sterling Heights, MI). For histological examination of kidneys, paraffin-embedded tissue samples were cut into 3-μm-thick sections and stained with H&E, periodic acid-Schiff, or Masson's trichrome (American MasterTeck, Lodi, CA). Slides were examined and scored for abnormalities by two nephropathologists. For detection of tubular necrosis, the scoring range from 0 to 3 was used to define noticeable cell damage in the form of hydropic change, cast formation, necrosis or apoptosis in the tissue area. The scoring criteria were defined as follows: score 0 = no noticeable cell damage; 1 = noticeable cell damage in tissue area 50%. Additional 3 μm-thick periodic acid-Schiff-stained paraffin sections of kidneys were evaluated for diabetic nephropathy from 4 different groups (n = 3 in each group): controls (m), diabetic (db), stem cell transplantation from control mice (Tx M) and from db mice (Tx db). Slides were examined by Olympus BX41 microscope under ×40 magnification. All glomeruli were counted in a single cross section as well as the numbers of lesioned microvessels, which were expressed as per 100 glomeruli. The normal mesangial area in the control animals was assigned 0 score, with scores 1+ if these were twice the size of the control, 2+ and 3+ when expanded to 3 and 4 times, respectively. Bone marrow (BM)-derived mononuclear cells were analyzed for an array of markers, including FITC or phycoerythrin (PE) conjugated anti-mouse CD117 (c-kit), CD150, sca-1, c-kit, CD34, CD31, CD44, CD45, flk,−1, and unconjucated anti-mouse vimentin, and nestin that paired with corresponding fluorescent secondary antibody (Jackson ImmunoResearch Laboratories). All primary antibodies were produced by BD Biosciences (Rockville, MD). Data were acquired using a FACScan cytometer equipped with a 488-nm argon laser and a 620-nm red diode laser and analyzed using CellQuest software (Becton Dickinson, San Jose, CA). The setup of FACScan was performed using unstained and single antibody-stained cells. To isolate MSCs from the bone marrow of db/db and db/m mice, the fresh BMDC preparations were re-suspended in complete MSC culture medium (StemCell Technologies Inc, Canada) and seeded into 6-well plates. The cells were than kept 3 days at 37°C in a CO2 incubator, fresh medium was changed, and the adherent layer was re-fed at 7 days. For analysis of apoptosis cells from 1 to 2 passages were used. To isolate endothelial progenitor cells (EPCs), BMDC were re-suspended in mouse EPC medium (Celprogen, San Pedro, CA) supplemented with 10% fetal bovine serum. Seven days after initiation of cultures on 4-well chamber slides (Nalge Nunc International) coated with Vitronectin (10 μg/ml), EPCs were assayed by costaining with acetylated LDL (acLDL)-Dil (Biomedical Technologies) for 3 hours at 37°C and FITC-conjugated Ulex europeaus Lectin (Sigma) for 30 minutes at 37°C, both characteristically staining cells of endothelial lineage.17Asahara T Takahashi T Masuda H Kalka C Chen D Iwaguro H Inai Y Silver M Isner JM VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.EMBO J. 1999; 18: 3964-3972Crossref PubMed Scopus (1692) Google Scholar Double-positive cells were counted as EPC in eight randomly selected fields of each slide. The proportion of apoptotic cells under basal and oxidative stress conditions, as detailed in Results, was examined using annexin V (BD pharmingen) and activated caspases detection using FITC-VAD-FMK (Calbiochem, La Jolla, CA). Results were summarized from three independent BM transfusion experiment and the numbers of mice for each study group were totaled: db/m n = 24, db/db control mice n = 20, dbCM n = 4, dbEbs-in vivo n = 5, dbTxm mice n = 10, dbTxdb mice n = 9, TxdbCM n = 4, TxdbEbs-in vivo n = 5, and TxdbEbs-ex vivo n = 5. The results were expressed as means ± SD The means of two populations were compared by Student's t-test. For multiple comparisons analysis of variance was used. Differences were considered significant at P < 0.05. We have previously demonstrated that conspicuous defects in endothelium-dependent vasorelaxation of aortic rings in db/db mice could be detected as early as 9 to 10 weeks of age, ie, just 2 weeks after establishment of a persistent hyperglycemia,14Brodsky SV Gealekman O Chen J Zhang F Togashi N Crabtree M Gross SS Nasjletti A Goligorsky MS Prevention and reversal of premature endothelial cell senescence and vasculopathy in obesity-induced diabetes by ebselen.Circ Res. 2004; 94: 377-384Crossref PubMed Scopus (181) Google Scholar in association with the increased numbers of prematurely senescent endothelial cells. Therefore, we pursued these studies of the macrovascular dysfunction in db/db mice recipients of BMDC transplants using acetylcholine-induced vasorelaxation assay. Aortic rings obtained from db/db mice showed a profound impairment of relaxation in response to the application of acetylcholine (Figure 1A). Maximal concentration of acetylcholine (100 μmol/L) elicited only a 29% relaxation of aortic rings compared with db/m mice. In contrast, db/db mouse-recipients of BMDC from db/db donors (dbTxdb group) showed a mild-to-moderate improvement of aortic relaxation (maximal relaxation of 56%), whereas the db/db mice receiving BMDC from their db/m littermates (dbTxm group) exhibited a dramatic improvement of aortic vasorelaxation with the maximal values achieving 81% of control db/m mice. Notably, all vessels responded to nitric oxide (NO) donor NONOate with equal relaxation (Figure 1B), thus indicating that the impaired responses to acetylcholine were due to defective endothelium-dependent relaxation. Monitoring blood glucose levels in db/db recipients of BMDC infusions (10 days after each infusion and 20 days after the last infusion) indicated a significant improvement of fasting blood glucose level in recipients of BMDC from db/m donors (dbTxm group) (Figure 2A). In mice that received BMDC from db/db donors (dbTxdb group), the improvement in fasting blood glucose level was transient and occurred only after the first transfusion, when the BMDC donor db/db mice were 8 weeks old (this is the age when hyperglycemia commences in db/db mice). Hyperglycemia resumed after the subsequent transfusions and was indistinguishable from non-treated db/db mice of equivalent age. Treatment of db/db mice with BMDC of either origin did not affect their body weight and all animals remained equally obese (Figure 2B). The results of the insulin tolerance test performed 20 days after the last BM transfusion showed that the sensitivity of mice to the injected insulin in the dbTxm group was improved compared with db/db control and dbTxdb group, although hypoglycemic responses remained equally delayed (Figure 2C), suggesting that the observed improvement of fasting blood glucose level in db/db recipients of BMDC from db/m donor mice was secondary to improved insulin sensitivity rather than reduced obesity. To further address the possibility that BM adoptive transfer can improve insulin sensitivity in the recipient diabetic mice, we measured their fasting plasma insulin, amylin, and glucagon level. Compared with db/db mice that exhibited elevated plasma insulin and amylin level, mice in the dbTxm group showed normalization of hyperinsulinemia and hyperamylinemia, whereas dbTxdb mice showed no improvement in insulin and only a partial improvement in amylin level (Figure 3, A and B). In accord with these results, the calculated glucose/insulin ratio and HOMA index showed significant improvement in the dbTxm group compared with db/db control and dbTxdb mice (Figure 3, C and D). Plasma glucagon measurements showed no significant differences between all of the experimental groups (Figure 3E). In aggregate, having excluded the contribution of other factors to the improved insulin sensitivity, the most plausible explanation is found in the BMDC-induced alleviation of endothelial dysfunction and improvement of microcirculation. Another possible explanation for the observed improved insulin sensitivity was related to the modulation of pro-inflammatory mediators.18Kahn SE Hull RL Utzschneider KM Mechanisms linking obesity to insulin resistance and type 2 diabetes.Nature. 2006; 444: 840-846Crossref PubMed Scopus (3706) Google Scholar To explore this possibility we measured the plasma concentration of 14 pro- and anti-inflammatory cyto- and chemokines (IL-1α and β, IL-6, IL-9, IL-10, IFNγ, IP-10, G-CSF, GM-CSF, TNFα, KC, MCP-1, MIP-1α, and mRANTES) following BM transfusion. The results showed elevated levels of IL-1α and G-CSF after BM transfusion regardless of the donors, elevated levels of IP-10 in dbTxm and elevated IL-10 levels in recipients of db/db BMDC (supplemental Figure 1, see http://ajp.amjpathol.org). In addition, we analyzed the extent of macrophage/mononuclear infiltration of the pancreatic and kidney parenchyma (supplemental Figure 2A, see http://ajp.amjpathol.org). Immunohistochemical staining showed that the CD68-positive cells were rare and scattered evenly in the pancreas and kidney sections with no differences detectable among the studied groups (supplemental Figure 2B–C, see http://ajp.amjpathol.org). The above analyses indicated the existence of low-grade pro-inflammatory conditions following the infusion of BMDC. These data ruled out the possibility that the improved insulin sensitivity following BM transfusion was due to the improved profile of pro-inflammatory cytokines. To examine the possibility of trans-differentiation of engrafted donor BMDC to insulin-producing cells and evaluate its contribution to the observed benefits following BM transfusion, we studied frozen sections of the pancreas. Fluorescent microscopy confirmed the presence of CM-Dil positive donor BMDC in the recipient's pancreas (supplemental Figure 2D, see http://ajp.amjpathol.org). These scattered cell tracker-labeled cells were rare and showed no difference in frequency between dbTxm and dbTxdb group. Examination of the pancreas co-stained with anti-mouse insulin antibody failed to show insulin-positive staining of engrafted donor BMDC (data not show). Immunohistochemical staining of insulin showed comparable density and normal morphology of the islets among db/m, db/db, and BMDC-treated diabetic mice (data not show). Quantification of the islets showed equal levels among different experimental groups (supplemental Figure 2E, see http://ajp.amjpathol.org). Our results suggested that a direct trans-differentiation to insulin-producing cell was not evident in the pancreas, and BMDC transfusion did not influence pancreatic islands structure and density. Effects of BMDC adoptive transfer on renal function in db/db mice were examined. The recipients of BMDC from db/m donors exhibited improved renal function (Figure 4, A and B) judging from urinary protein/creatinine ratio and plasma creatinine level. Glomeruli showed mild to moderate hypertrophy in all db/db animals compared with controls and in addition displayed mesangial expansion (range, 2 to 2.5; average 2.3+) and arteriolar hyalinosis (range, 8% to 18%; average 9%; supplemental Figure 3, see http://ajp.amjpathol.org). An occasional lesion of focal and segmental glomerulosclerosis, containing extracellular lipid droplets, was also evident (1/3 animals) in db/db mice. Kidneys from dbTxm group showed mild decrease in mesangial expansion (range 1.5 to 2.0; average 1.66+) and in areteriolar hyalinosis (range, 2% to 4%; average 3.3%). No lesions of focal and segmental glomerulosclerosis were present in any of these sections obtained from dbTxm mice. The decline in mesangial expansion (range, 2 to 2.5; average 2.17+) and in areteriolar hyalinosis (range, 2% to 7%; average 4.66%) were less impressive in the dbTxdb animals. An occasional lesion of focal and segmental glomerulosclerosis was evident in one of the three animals as well. Of note, these changes were not universal and analysis showed that there was a subgroup, which was analyzed separately (40%), that showed the deterioration of proteinuria and plasma creatinine level after BM transfusion from either donor group. (Figure 4, C and D). Consistent with these findings, histological examination of kidney sections revealed elevated pathological score of tubular necrosis in this subgroup along with an increased glomerular volume (Figure 4E). These findings are not inconsistent with multiple clinical observations that pointed out the risk of acute kidney injury in 53% to 92% after BM transplantation.19Zager RA O'Quigley J Zager BK Alpers CE Shulman HM Gamelin LM Stewart P Thomas ED Acute renal failure following bone marrow transplantation: a retrospective study of 272 patients.Am J Kidney Dis. 1989; 13: 210-216PubMed Scopus (228) Google Scholar, 20Zager RA Acute renal failure in the setting of bone marrow transplantation.Kidney Int. 1994; 46: 1443-1458Crossref PubMed Scopus (153) Google Scholar, 21Parikh CR McSweeney PA Korular D Ecder T Merouani A Tay
Referência(s)