Diabetes Mellitus Induces Bone Marrow Microangiopathy
Objective— The impact of diabetes on the bone marrow (BM) microenvironment was not adequately explored. We investigated whether diabetes induces microvascular remodeling with negative consequence for BM homeostasis.
Methods and Results— We found profound structural alterations in BM from mice with type 1 diabetes with depletion of the hematopoietic component and fatty degeneration. Blood flow (fluorescent microspheres) and microvascular density (immunohistochemistry) were remarkably reduced. Flow cytometry verified the depletion of MECA-32+ endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker β-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage− c-Kit+ Sca-1+ cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage− c-Kit+ Sca-1+ cells predominantly in the low-perfused part of the marrow. Cell depletion was associated to increased oxidative stress, DNA damage, and activation of apoptosis. Boosting the antioxidative pentose phosphate pathway by benfotiamine supplementation prevented microangiopathy, hypoperfusion, and lineage− c-Kit+ Sca-1+ cell depletion.
Conclusion— We provide novel evidence for the presence of microangiopathy impinging on the integrity of diabetic BM. These discoveries offer the framework for mechanistic solutions of BM dysfunction in diabetes.
Diabetic patients have ischemic complications more frequently than nondiabetic subjects and also show a worse clinical outcome after an ischemic event. This prognostic disadvantage is partly dependent on diabetes-induced impairment of reparative angiogenesis. The contribution of circulating cells in maintenance of vascular integrity and recovery from ischemic complications has been also acknowledged. Tissue injury triggers the bone marrow (BM) to release progenitor cells (PC) and monocytes with proangiogenic capacities into the peripheral circulation.1–3 A default version of this cellular response may account for the weakened healing capacity in diabetes. However, whether diabetes may damage stem cells (SC) inside the BM either directly or by altering their microenvironment remains to be elucidated.
Maintenance of BM homeostasis is dependent on the interaction between SC and cells of the supportive microenvironment, where SC self-renew, differentiate, or die. Regulatory components of the niche include endothelial cells (EC), mesenchymal cells, and adipocytes. The cellular composition and location of the niche are associated with specialized functions. For instance, the vascular niche, composed of lineage-committed PC, mature hematopoietic cells, stromal cells, and cells of the fenestrated sinusoidal endothelium, preside over the trafficking of cells and solutes between the marrow and circulation.4 The osteoblastic niche, located near the endosteal bone and its trabecular projections, is regarded as the main repository of primitive SC of the marrow.5 The low-oxygenated osteoblastic microenvironment is ideal to maintenance of SC quiescence, with SC differentiation occurring along the oxygen ascent toward the vasculature.6,7 However, some endosteal niches are well-perfused, being enmeshed in microvessels that penetrate the bone, and are thereby equally influenced by signals from osteoblasts and EC and by chemical cues from the circulation.8 Furthermore, SC scattered between the 2 main niches may represent transition entities moving back and forward between the endosteum and vasculature.9
In this study we investigated the status of vascular cells, hematopoietic cells, and their niches in BM of diabetic mice. Results show profound marrow remodeling with depletion of the hematopoietic component and presence of a so-far-unreported form of microangiopathy. Importantly, cell depletion more prominently affected the osteoblastic niche because of the generation of a steeper perfusion gradient across the marrow. Inhibition of oxidative stress prevented BM microangiopathy, hypoperfusion, and hematopoietic cell depletion.
Materials and Methods
A detailed, expanded Materials Methods section is available (available online at http://atvb.ahajournals.org).
Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (the Institute of Laboratory Animal Resources, 1996) and with approval of the British Home Office. Type 1 diabetes (T1D) was induced in male CD1 mice (Charles River, Margate, Kent, United Kingdom) by streptozotocin.10 Age-matched male CD1 mice injected with the vehicle of streptozotocin served as controls. Diabetes was assessed by measurement of glycemia at fast and glycosuria.
At 4 weeks from diabetes induction, T1D subgroups were randomly assigned to receive benfotiamine (BFT; 70 mg/kg body weight per day) or vehicle (1 mmol/L HCl) in drinking water for 24 weeks. Nondiabetic age-matched vehicle-treated male mice served as controls.
Measurement of Marrow Blood Flow
Blood flow was assessed by fluorescent microspheres.
Bone Fixation, Decalcification, and Sectioning
Bones were cleaned from muscle and connective tissue, fixed, decalcified, and finally processed for paraffin embedding.
Total volume of the marrow was computed from longitudinal and cross BM sections on an Olympus BX40 microscope. Giemsa, Trichrome Masson, and Gomori staining were performed to identify the structural composition of BM.
To determine capillary and sinusoid density, BM sections were stained with Isolectin IB4 (endothelial marker). Capillaries were recognized as small, regular endothelial structures whose lumen-size does not exceed the diameter of an erythrocyte, whereas sinusoids were identified as irregular vessels, lined by a thin layer of Isolectin IB4-positive EC, and able to contain several erythrocytes (Supplementary Figure I, available online at http://atvb.ahajournals.org). Arterioles were recognized by the vascular smooth muscle cell marker α-smooth muscle actin and Isolectin IB4. The number of capillaries, sinusoids, and arterioles was counted through the entire area of marrow and expressed as average density per mm2 of tissue. Additionally, vascular endothelial (VE)-cadherin-2 was used to visualize vascular niches. The endosteal surface lined by osteoblasts was visualized by an anti-neural-cadherin antibody.11 Mouse c-Kit and Sca-1 antigens were used to identify hematopoietic PC, and Ter119 was used to identify erythroid cells. DNA damage was assessed by staining for p-H2AX.12 A list of used antibodies is reported in Supplementary Table I (available online at http://atvb.ahajournals.org).
Selection of BM EC
Freshly harvested BM cells were immunomagnetically depleted of CD11b-expressing cells to eliminate myeloid/monocyte fraction and cultured on 0.1% gelatin in DMEM 20% fetal bovine serum supplemented with AcSDKP to avoid SC and fibroblasts contamination.13 When confluent, cells were analyzed by flow cytometry and immunocytochemistry to assess the expression of endothelium-specific markers. Using the same isolation protocol, confluent BM EC were used in functional studies.
Functional and Western Blot Assays on BM EC
Cell senescence was assessed by measuring β-Gal activity and reactive oxygen species (ROS) using MitoTracker Red CM-H2XROS probe. Migration was assayed using a 24-well transwell set-up and in vitro network formation on matrigel.14 For static adhesion, BM EC were cultured to confluence on 0.1% gelatin-coated glass covers and treated overnight with tumor necrosis factor-α (10 ng/mL). Next, BM mononuclear cells (MNC) from controls mice were prelabeled with calcein-AM, resulting in green fluorescence, and allowed to adhere for 30 minutes on BM EC. Samples were then washed and adherent BM MNC were counted using confocal fluorescent microscopy. To study the influence of flow, confluent BM EC were stimulated as described and mounted onto the microscope stage using a perfusion, open and closed mini chamber system (LaCon) and connected to a perfusion pump. Adhesion was visualized by phase-contrast microscopy and recorded in real time. Transendothelial electric resistance was evaluated by electric cell-substrate impedance sensing. To study transendothelial migration of BM MNC prepared from controls or T1D mice, cells were prelabeled with PKH67 (Sigma) and then left to migrate toward SDF-1 or vehicle through BM EC monolayers on coated transwell filters. Finally, protein expression of phosphorylated VE-cadherin and protein tyrosine kinase 2 in BM EC was measured by Western blot.
Isolation of Marrow Cells From Trabecular Bone
Hematopoietic stem cell isolation kit (Millipore UK) was used for isolation of marrow cells from trabecular bone.
Colony-Forming Cell Assay
Freshly harvested BM cells from trabecular bone were seeded on methylcellulose (1×104 cells/dish) and cultured for 14 days before scoring colonies.
Flow Cytometry Analysis
Freshly harvested BM cells were washed with ice-cold Hank balanced salt solution containing 0.5% bovine serum albumin and 0.02% sodium azide. BM cells were then stained in the same buffer with anti-lineage mixture (Alexa 488), anti-Sca-1 (phycoerythrin), anti-CD34 (Alexa 647), and antic-Kit (Alexa 750 or Alexa 647 when CD34 was omitted). To recognize EC, BM cells were stained with anti-MECA-32 (Biotin), followed by streptavidin-allophycocyanin conjugate. To detect apoptosis, BM cells were stained with annexin V (fluorescein isothiocyanate). ROS-positive cells were identified using CM-H2DCFDA. Distribution of BM cells according to BM perfusion gradient was evaluated using the Hoechst 33342 (Hoe) dye.7 Briefly, Hoe was injected through the tail vein and the animals were euthanized 10 minutes later to collect the hind limb BM. Cells in microenvironments that are well-perfused by blood are those exposed to the highest concentrations of Hoe, whereas cells in microenvironments that are less perfused are exposed to lower concentrations of Hoe. Flow cytometry identification of cells stained high or low with Hoe (Hoehigh and Hoelow, respectively) allowed for recognition of cell distribution in high-perfused vs low-perfused regions of BM (Supplementary Figure II). Flow cytometry was performed on FACSCanto II and FACSLSRII (BD Biosciences) equipped with FACSDiva software (BD Biosciences). Data were represented using “logical” displays. A list of used antibodies is reported in Supplementary Table II.
Differences between multiple groups were compared by analysis of variance, followed by a Holm-Sidak multiple comparison test. Two-group analysis was performed by t test (paired or unpaired as appropriate). P<0.05 was considered significant.
Diabetes Reduces BM Volume and Cellularity
First, we compared the BM structure of T1D mice at 27 to 30 weeks from the onset of diabetes to age-matched nondiabetic controls. Diabetes remarkably reduced the hematopoietic fraction and caused fat accumulation and osteopenia (Figure 1). No structural alteration was observed at 10 days after diabetes induction (data not shown), discounting an acute toxic effect of streptozotocin on the BM.
Microangiopathy in Diabetic BM
Cumulative vascular density was reduced by 2.9-fold in BM of T1D mice (P<0.001 vs controls). Analysis of perfused vessels, identified by binding of intracardially injected isolectin IB4, revealed a consistent reduction of sinusoids, capillaries, and arterioles. Furthermore, the microvasculature appeared fragmented with bleeding into the surrounding marrow (Figure 2A–D).
Flow cytometry analysis of BM single-cell suspensions, using an antibody specific for the EC marker MECA-32, confirmed BM EC depletion and increased BM EC apoptosis in diabetes (Figure 2E, F).
Functional Alterations of Diabetic BM EC
BM EC were isolated from T1D and control mice and their purity was confirmed by flow cytometry and immunocytochemistry (Supplementary Figure III). We found that T1D BM EC express higher levels of mitochondrial ROS (Figure 3A) and cell senescence marker β-galactosidase (Figure 3B), are unresponsive to chemoattractant stimuli, like SDF-1 and vascular endothelial growth factor A (Figure 3C), and fail to form network structures on matrigel (Figure 3D). Furthermore, we observed an increased adhesion of BM MNC to T1D BM EC under static conditions and after introduction of shear flow (Figure 3E, F).
Another hallmark of diabetic microvasculature is its augmented permeability. Confluent T1D BM EC showed a 14%±2% reduction in transendothelial resistance compared to controls BM EC (P<0.05), which was abrogated by the ROS scavenger N-Acetyl-cysteine, pinpointing oxidative stress as a determinant of altered cell–cell interaction. ROS facilitates transendothelial migration of BM-derived PC through phosphorylation of VE-cadherin by the redox-sensitive protein tyrosine kinase 2.15,16 We found that T1D BM EC have higher phosphorylation levels of VE-cadherin (at tyrosine 731, the β-catenin binding site) and protein tyrosine kinase 2 (at tyrosine 402, which is the autophosphorylation site for protein tyrosine kinase 2) compared with control BM EC (Figure 3G). Furthermore, T1D BM MNC transmigrate as efficiently as control BM MNC in the presence of nondiabetic endothelium (Figure 3H, left). In contrast, nonspecific migration of BM MNC was enhanced and SDF-1–stimulated migration was abolished in the presence of diabetic endothelium, thus suggesting endothelial barrier dysfunction in T1D (Figure 3H, right).
Diabetes Causes Depletion of BM Sca-1posc-Kitpos Cells
Immunohistochemical analysis documented the reduction of Sca-1posc-Kitpos (SK) cells in BM of T1D, especially at the level of the osteoblastic niche, identified by staining osteoblast lining with neural-cadherin (Figure 4, Supplementary Figure IV). Furthermore, considering longitudinal and coronal sections of BM, we verified that the distance of SK cell clusters of the osteoblastic niche to sinusoids is longer in marrow of T1D mice (9.0±0.4 cell diameters) compared to that in controls (5.5±0.4 cell diameters; P<0.001).
Flow cytometry analysis confirmed the effect of diabetes on reducing the relative frequency of Lineage− SK (LSK) cells in marrow of the femoral cavity or trabecular bone, a porous plexus enriched with SC and osteoblasts (Figure 5A).5 We also found that the subfraction of primitive CD34−LSK cells is remarkably reduced in T1D marrow (3.6±0.7 per 100 000 BM cells) compared to controls (27.0±3.0 per 100 000 BM cells; P<0.01). Concordantly, colony-forming-unit assays showed a reduced formation of multipotent PC colonies (colony forming unit granulocyte-erythroid-makrophage-megakaryocyte [c.f.u. GEMM]) by trabecular BM cells of T1D mice (Figure 5B). However, the colony-forming activity of lineage-committed PC was similar in diabetic and control mice, suggesting compensation downstream to multipotent PC.
Diabetes Reduces BM Perfusion
T1D mice showed a remarkably reduced BM perfusion at the level of femur (0.17±0.01 vs 0.27±0.02 mL/min per gram in controls; P<0.01) and tibia (0.11±0.01 vs 0.18±0.03 mL/min per gram in controls; P<0.01).
Predominant LSK Cell Depletion in the Hypoperfused Part of the Marrow
We then determined the relative position of LSK cells with respect to in vivo Hoe dye perfusion gradient.7 Hoe was injected intravenously and then the degree of uptake of the dye by BM cells from different locations was evaluated by flow cytometry. We found that 53% of total LSK cells are located in the Hoelow perfusion region of controls BM, but this fraction decreased to 21% in T1D BM (Figure 6A, central panel). Reversing the gating procedure, we analyzed the abundance of LSK cells in total cells and lymphomonocyte fraction of each Hoe perfusion area (Figure 6B). Results confirmed the selective depletion of LSK cells of the low-perfused zone of T1D BM, whereas the high-perfused zone, which corresponds to the predominant localization of MECA-32pos BM EC (eg, the vascular niche), was relatively preserved. MECA32+ EC were reduced overall in T1D BM (Figure 6C) and, considering their relative distribution, also shifted from the low to the high Hoe perfusion area (Figure 6A, right).
Increased Oxidative Stress in Diabetic BM
Next, we measured levels of oxidative stress in BM cells using CM-H2DCFDA, a cell-permeable intracellular ROS indicator. Flow cytometry analysis showed that ROShigh SK cells are greatly increased in T1D BM (Figure 7A). We also verified the presence of higher mitochondrial ROS levels in BM MNC from T1D trabecular marrow using MitoTracker Red CM-H2XROS (Figure 7B).
Excessive oxidative stress reportedly causes DNA damage and reduces the lifespan of BM SC.17 Levels of p-H2AX (Ser139), a marker of double DNA strand breaks, were 2.5-fold higher in T1D BM cells compared to controls (Figure 7C). Because H2AX is phosphorylated by ataxia telangiectasia mutated, we analyzed ataxia telangiectasia mutated expression by quantitative polymerase chain reaction and found it 2.6-fold higher in T1D BM cells compared to controls. Furthermore, flow cytometry analysis of Annexin V-positive cells unraveled the increased apoptosis of SK cells from BM of T1D mice (Figure 7D).
Stimulation of Antioxidative Mechanism Prevents Microangiopathy and LSK Cell Depletion
We found that diabetes reduces the activity of transketolase and G6PDH, the rate-limiting enzymes of the pentose phosphate pathway, which represents a fundamental source of antioxidant equivalents and substrates for DNA synthesis and repair (Figure 8A, B).
We then asked whether activation of this antioxidative mechanism may protect BM from diabetes-induced damage. Boosting the thiamine-dependent enzyme transketolase by BFT supplementation (Figure 8A) restored G6PDH activity (Figure 8B) and prevented microangiopathy (Figure 8C) and hypoperfusion of diabetic BM (Figure 8D). Furthermore, BFT prevented oxidative stress (Figure 8E) and p-H2AX elevation (Figure 8F) in T1D BM cells. Importantly, these effects of BFT were associated to prevention of LSK cell depletion, both in terms of absolute number (Figure 8G) and relative proportion to total BM cells (Figure 8H), and inhibition of apoptosis (Figure 8I). Analysis of cell distribution across the Hoe perfusion gradient confirmed the protective action of BFT against diabetes-induced LSK cell depletion (Figure 8J, K).
Here we show for the first time to our knowledge the presence of diabetic microangiopathy altering the marrow milieu. Microvascular rarefaction was associated with endothelial dysfunction, encompassing reduced migratory capacity, impaired angiogenic activity, increased adhesiveness, and endothelial barrier disruption. Importantly, these defects were observed after culturing diabetic BM EC in normal glucose, in line with the recent demonstration of epigenetic changes caused by transient hyperglycemia.18
Previous studies have documented the important role of the BM endothelium in maintenance of marrow homeostasis through paracrine and physical interaction with other cells of the marrow.19,20 Another important function of BM vasculature is to deliver nutrients and oxygen to marrow cells. The peculiar distribution of microvasculature creates differentially perfused environments across the marrow. The most primitive stem cells are believed to reside in the osteoblastic niche at the lowest end of the physiological perfusion gradient, protected from oxidative stress.6,7 However, recent studies demonstrated that a large fraction of endosteal stem cells is enmeshed in vessel networks.21 In diabetic BM, the ongoing microvascular rarefaction inevitably alters the path-length for oxygen and nutrient diffusion, and, as a consequence, an increasing fraction of marrow becomes critically hypoperfused and secluded from the influence of the vascular niche. Our results indicate that LSK cells of the osteoblastic niche can barely survive in such a harsh environment. However, the BM vasculature can offer an ultimate shelter, as documented by the relative conservation of LSK cells in the perivascular space. To the best of our knowledge, the only precedent for marrow cell depletion in the hypoxic microenvironment, often identified with the osteoblastic niche, is represented by the hematopoietic decline described in aging rodents.6 The model of accelerated senescence fits well with diabetic BM remodeling, because in both conditions fat accumulation occurs along with osteopenia. The mechanism that underpins aging-induced and diabetes-induced increases in adipocyte abundance remains unknown. Fat accumulation could serve not only to fill the empty marrow, pushing marrow cells toward the vasculature, but also to participate in the ongoing diabetic remodeling by secreting paracrine factors and proinflammatory cytokines.22 Of note, a similar remodeling was observed in obese leptin-receptor mutant mice, a model of insulin-resistant type 2 diabetes (P. Madeddu, unpublished observations, 2009).
The physiological gradient of ROS acts as a signaling mechanism governing functional compartmentalization of stem cells. Those precious cells, necessary for regeneration of almost all the rest of the whole organism, reside in the “low-risk zone,” ideal for maintenance of quiescence. The function of the ROShigh zone adjacent to the marrow vasculature instead is to facilitate stem cell maturation.6 Under pathological conditions, however, excessive production of ROS might endanger the viability of stem cells. Genetically modified mice, lacking essential components of the regulatory system that maintain ROS within the physiological range, show accelerated stem cell senescence and progressive bone marrow failure,23–25 replicating the situation observed in mice exposed to the oxidant buthionine sulfoxime.17 Our data show that an elevation in intracellular ROS infringes on DNA integrity and compromises marrow cell function in a model of common human disease. Different mechanisms might contribute to increasing oxidative stress in LSK cells, including critical hypoperfusion and high glucose, which are both potent activators of ROS generation by mitochondrial complex III.26,27 In addition, transition metal iron from extravasated erythrocytes can be a potent source of ROS via the Fenton reaction. Another mechanism consists of the reduced activity of antioxidative mechanisms, such as the pentose phosphate pathway. In line with the latter, benfotiamine buffered the diabetes-induced disruptive effect on LSK cells.
The extensive remodeling of bone marrow observed in diabetic mice may not inspire therapeutic optimism. However, previous studies showed that glucose-lowering therapies can restore progenitor cell function to some extent.28 Similarly, in genetically modified animals unable to modulate ROS production, antioxidant administration restored the reconstitutive capacity of hematopoietic stem cells, thereby preventing bone marrow failure.23,24 Our study newly shows that benfotiamine stimulates antioxidative defense through activation of transketolase and protects vascular and LSK cells from oxidative stress and apoptosis.
In conclusion, our results demonstrate the deleterious effect of diabetes on bone marrow homeostasis. Our characterization of the molecular and cellular signature of diabetic pathology in bone marrow along with successful results of BFT treatment may lead to beneficial therapies for human disease. Whether thiamine derivatives may clinically reverse BM failure in diabetes represents the objective of future investigation.
Sources of Funding
Wellcome Trust (083018/Z/07/Z), BHF (PG/06/096/21325, FS/06/083/21828), and EC-FP7–53861 to P.M.; the Dutch Heart Foundation (2005T039) and NWO Veni grant (916.76.053) to J.D.v.B. and F.P.J.v.A.; BIOSCENT FP7-NMP-214539, MIUR grant (AL2YNC), and THEAPPL to F.Q. C.E. holds a BHF Basic Science fellowship (BS/05/01).
A.O. and M.S. contributed equally to the study.
Received November 9, 2009; revision accepted December 21, 2009.
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