Aberrant bone marrow-derived microglia in the hypothalamus may dysregulate appetite in diabetes.

Bone marrow derived cells (BMDCs) migrate into the hypothalamus, where those cells give rise to microglia to regulate food intake. Given the fact that diabetes functionally impairs BMDCs, we hypothesized that diabetic microglia would fail to exhibit physiological function, accounting for hyperphagia in diabetes. To examine the role of BMDCs, total bone marrow cells from GFP transgenic mice were transplanted into wild type mice in which diabetes was induced by streptozotocin. We first confirmed that bone marrow transplantation could be utilized to examine BMDCs in the brain parenchyma as GFP positive cells could engraft the brain parenchyma and give rise to microglia even when the BBB was intact in the recipient mice. While diabetic mice manifested hyperphagia, BMDCs were in smaller number in the hypothalamus with less response to fasting in the brain parenchyma compared to nondiabetic mice. This finding was also confirmed by examining nondiabetic chimera mice in which BMDCs were diabetic. Those mice also exhibited less response of BMDCs in response to fasting. In conclusion, diabetic BMDCs had less response of microglia to fasting, perhaps accounting for diabetic hyperphagia.

Complete remission of diabetes with a transient HDAC inhibitor and insulin in streptozotocin mice.

Despite the growing epidemic worldwide, diabetes is an incurable disease. We have been focusing on why diabetes manifests refractoriness to any therapy. We recently found that abnormal bone marrow-derived cells (BMDCs), namely, Vcam-1+ST-HSCs, was a key mechanism for diabetic complications. We then hypothesize that those aberrant BMDCs sustainedly impair pancreatic ß cells. Here we show that eliminating abnormal BMDCs using bone marrow transplantation results in controlling serum glucose in diabetic mice, in which normoglycemia is sustained even after cessation of insulin therapy. Alternatively, abnormal BMDCs exhibiting epigenetic alterations are treated with an HDAC inhibitor, givinostat, in diabetic mice. As a result, those mice are normoglycemic along with restored insulin secretion even following the cessation of both insulin and givinostat. Diabetic cell fusion between abnormal BMDCs and resident cells is significantly blocked by the combination therapy in the pancreatic islets and thymus while surgical ablation of the thymus completely eliminates therapeutic protection in diabetic mice. In conclusion, diabetes is an epigenetic stem cell disorder with thymic disturbances. The combination may be applied to patients aiming at complete remission from diabetes in clinical medicine.

Bone marrow-derived vasculogenesis leads to scarless regeneration in deep wounds with periosteal defects.

Deep skin wounds with periosteal defects, frequently caused by traffic accidents or radical dissection, are refractory. Transplant surgery is frequently performed, but patients are subjected to stress for long operation periods, the sacrifice of donor regions, or several complications, such as flap necrosis or intractable ulcers. Even if the defects are covered, a scar composed of fibrous tissue remains in the body, which can cause itching, dysesthesia, or repeated ulcers because of the lack of distribution of peripheral nerves or hair follicles. Thus, treatments with the aim of regenerating lost tissue for deep wounds with periosteal defects are needed. Here, we show that the use of gelatin sponges (GS), which have been used as haemostatic materials in clinical practice, allowed the regeneration of heterogeneous tissues, including periosteum, skin, and skin appendages, when used as scaffolds in deep wounds with periosteal defects in rats. Bone marrow transplantation in rats revealed the mechanism by which the microenvironment provided by GS enabled bone marrow-derived cells (BMDCs) to form a vascular niche, followed by regeneration of the periosteum, skin, or skin appendages such as hair follicles by local cells. Our findings demonstrated that vascular niche formation provided by BMDCs is crucial for heterogeneous tissue regeneration.

Inhibition of mitochondrial fission protects podocytes from albumin-induced cell damage in diabetic kidney disease.

AIMS: Identifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD. METHODS: Diabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx. RESULTS: Neuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.

GLT1 gene delivery based on bone marrow-derived cells ameliorates motor function and survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is an intractable neurodegenerative disease. CD68-positive bone marrow (BM)-derived cells (BMDCs) accumulate in the pathological lesion in the SOD1(G93A) ALS mouse model after BM transplantation (BMT). Therefore, we investigated whether BMDCs can be applied as gene carriers for cell-based gene therapy by employing the accumulation of BMDCs. In ALS mice, YFP reporter signals were observed in 12-14% of white blood cells (WBCs) and in the spinal cord via transplantation of BM after lentiviral vector (LV) infection. After confirmation of gene transduction by LV with the CD68 promoter in 4-7% of WBCs and in the spinal cord of ALS mice, BM cells were infected with LVs expressing glutamate transporter (GLT) 1 that protects neurons from glutamate toxicity, driven by the CD68 promoter, which were transplanted into ALS mice. The treated mice showed improvement of motor behaviors and prolonged survival. Additionally, interleukin (IL)-1ß was significantly suppressed, and IL-4, arginase 1, and FIZZ were significantly increased in the mice. These results suggested that GLT1 expression by BMDCs improved the spinal cord environment. Therefore, our gene therapy strategy may be applied to treat neurodegenerative diseases such as ALS in which BMDCs accumulate in the pathological lesion by BMT.

Malfunctioning CD106-positive, short-term hematopoietic stem cells trigger diabetic neuropathy in mice by cell fusion.

Diabetic neuropathy is an incurable disease. We previously identified a mechanism by which aberrant bone marrow-derived cells (BMDCs) pathologically expressing proinsulin/TNF-α fuse with residential neurons to impair neuronal function. Here, we show that CD106-positive cells represent a significant fraction of short-term hematopoietic stem cells (ST-HSCs) that contribute to the development of diabetic neuropathy in mice. The important role for these cells is supported by the fact that transplantation of either whole HSCs or CD106-positive ST-HSCs from diabetic mice to non-diabetic mice produces diabetic neuronal dysfunction in the recipient mice via cell fusion. Furthermore, we show that transient episodic hyperglycemia produced by glucose injections leads to abnormal fusion of pathological ST-HSCs with residential neurons, reproducing neuropathy in nondiabetic mice. In conclusion, we have identified hyperglycemia-induced aberrant CD106-positive ST-HSCs underlie the development of diabetic neuropathy. Aberrant CD106-positive ST-HSCs constitute a novel therapeutic target for the treatment of diabetic neuropathy.

A novel role for bone marrow-derived cells to recover damaged keratinocytes from radiation-induced injury.

Exposure to moderate doses of ionizing radiation (IR), which is sufficient for causing skin injury, can occur during radiation therapy as well as in radiation accidents. Radiation-induced skin injury occasionally recovers, although its underlying mechanism remains unclear. Moderate-dose IR is frequently utilized for bone marrow transplantation in mice; therefore, this mouse model can help understand the mechanism. We had previously reported that bone marrow-derived cells (BMDCs) migrate to the epidermis-dermis junction in response to IR, although their role remains unknown. Here, we investigated the role of BMDCs in radiation-induced skin injury in BMT mice and observed that BMDCs contributed to skin recovery after IR-induced barrier dysfunction. One of the important mechanisms involved the action of CCL17 secreted by BMDCs on irradiated basal cells, leading to accelerated proliferation and recovery of apoptosis caused by IR. Our findings suggest that BMDCs are key players in IR-induced skin injury recovery.

Protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes.

To examine the cell-protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes, we analyzed the renal phenotype of tamoxifen (TM)-inducible podocyte-specific Atg5-deficient (iPodo-Atg5-/-) mice with experimental endothelial dysfunction. In both control and iPodo-Atg5-/- mice, high fat diet (HFD) feeding induced glomerular endothelial damage characterized by decreased urinary nitric oxide (NO) excretion, collapsed endothelial fenestrae, and reduced endothelial glycocalyx. HFD-fed control mice showed slight albuminuria and nearly normal podocyte morphology. In contrast, HFD-fed iPodo-Atg5-/- mice developed massive albuminuria accompanied by severe podocyte injury that was observed predominantly in podocytes adjacent to damaged endothelial cells by scanning electron microscopy. Although podocyte-specific autophagy deficiency did not affect endothelial NO synthase deficiency-associated albuminuria, it markedly exacerbated albuminuria and severe podocyte morphological damage when the damage was induced by intravenous neuraminidase injection to remove glycocalyx from the endothelial surface. Furthermore, endoplasmic reticulum stress was accelerated in podocytes of iPodo-Atg5-/- mice stimulated with neuraminidase, and treatment with molecular chaperone tauroursodeoxycholic acid improved neuraminidase-induced severe albuminuria and podocyte injury. In conclusion, podocyte autophagy plays a renoprotective role against diabetes-related structural endothelial damage, providing an additional insight into the pathogenesis of massive proteinuria in diabetic nephropathy.

Transplantation of M2-Deviated Microglia Promotes Recovery of Motor Function after Spinal Cord Injury in Mice.

Despite the poor prognosis of spinal cord injury (SCI), effective treatments are lacking. Diverse factors regulate SCI prognosis. In this regard, microglia play crucial roles depending on their phenotype. The M1 phenotype exacerbates neuroinflammation, whereas the M2 phenotype promotes tissue repair and provides anti-inflammatory effects. Therefore, we compared the effects of M2 and M1 microglia transplantation on SCI. First, we established a method for effective induction of M1 or M2 microglia by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-4, respectively, to be used for transplantation in a SCI mouse model. In the M2 microglia transplantation group, significant recovery of motor function was observed compared with the control and M1 groups. Elevated transcription of several neuroprotective molecules including mannose receptor C type 1 (Mrc1), arginase 1 (Arg1), and insulin-like growth factor 1 (Igf1) was observed. Moreover, intramuscular injection of FluoroRuby dye revealed recovery of retrograde axonal transport from the neuromuscular junction to upstream of the injured spinal cord only in the M2-transplanted group, although the number of migrated microglia were comparable in both M1 and M2 groups. In conclusion, our results indicated that M2 microglia obtained by IL-4 stimulation may be a promising candidate for cell transplantation therapy for SCI.

Advanced Technology for Gene Delivery with Homing Peptides to Spinal Cord through Systemic Circulation in Mice.

Homing peptides to the spinal cord were identified and isolated using phage display technology. In vivo biopanning was performed by intravenous systemic injection of a phage library to screen specific peptides targeting the spinal cord of mice. Analyses of the sequences of targeted phages yielded two candidate peptides targeting the spinal cord: SP1 (C-LHQSPHI-C) and SP2 (C-PTNNPRS-C). These peptides were synthesized and intravenously injected into mice to evaluate their tissue specificity and potential as gene delivery carriers. The complexes between SP1 or SP2 peptides and the plasmid vector expressing the reporter gene could induce gene transduction in the spinal cord through systemic injection without gene expression in the brain, liver, and kidney. In addition, intravenous injection of the complex between SP1 and the vectors induced interleukin-4 expression in the spinal cord, resulting in effective suppression of lipopolysaccharide-induced hyperalgesia. Therefore, intravenously administered spinal cord homing peptides complexed with a plasmid vector provided tissue-specific treatment featuring gene delivery to the CNS through systemic circulation. This novel method of gene delivery is feasible and has great potential for clinical application.

Stem cell factor induces polarization of microglia to the neuroprotective phenotype in vitro.

Microglia are classified mainly into the M1 or M2 phenotypes, which evoke either proinflammatory or neuroprotective responses. Given the association of microglia with the pathogenesis of neuronal diseases, they are in focus as therapeutic targets for the treatment of such conditions. Stem cell factor (SCF) is a ligand for the c-kit receptor, one of the differentiation factors for bone marrow cells. In this study, characteristics of SCF-activated microglia and their effects on neurons were analyzed to investigate the therapeutic potential of SCF in neuronal diseases. SCF was found to induce proliferation, migration, and phagocytosis of microglia. In addition, SCF-derived microglia showed a neuroprotective phenotype expressing anti-inflammatory cytokines, growth factors, and M2 markers as compared to the phenotype shown by granulocyte macrophage-colony stimulating factor-derived microglia expressing inflammatory cytokines and M1 markers. Furthermore, supernatant medium from SCF-activated microglia enhanced cell proliferation and protection from cell death in NSC-34 neuronal cells. We conclude that SCF modulates microglial functions and induces activation of the neuroprotective effects of microglia, which could be used for treatment of neuronal diseases.

Gene Therapy for Neuropathic Pain through siRNA-IRF5 Gene Delivery with Homing Peptides to Microglia.

Astrocyte- and microglia-targeting peptides were identified and isolated using phage display technology. A series of procedures, including three cycles of both in vivo and in vitro biopanning, was performed separately in astrocytes and in M1 or M2 microglia, yielding 50-58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocytes (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely, astrocytes, M1 microglia, or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between peptide MG1 and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells.

Hyperglycemia Induces Skin Barrier Dysfunctions with Impairment of Epidermal Integrity in Non-Wounded Skin of Type 1 Diabetic Mice.

Diabetes causes skin complications, including xerosis and foot ulcers. Ulcers complicated by infections exacerbate skin conditions, and in severe cases, limb/toe amputations are required to prevent the development of sepsis. Here, we hypothesize that hyperglycemia induces skin barrier dysfunction with alterations of epidermal integrity. The effects of hyperglycemia on the epidermis were examined in streptozotocin-induced diabetic mice with/without insulin therapy. The results showed that dye leakages were prominent, and transepidermal water loss after tape stripping was exacerbated in diabetic mice. These data indicate that hyperglycemia impaired skin barrier functions. Additionally, the distribution of the protein associated with the tight junction structure, tight junction protein-1 (ZO-1), was characterized by diffuse and significantly wider expression in the diabetic mice compared to that in the control mice. In turn, epidermal cell number was significantly reduced and basal cells were irregularly aligned with ultrastructural alterations in diabetic mice. In contrast, the number of corneocytes, namely, denucleated and terminally differentiated keratinocytes significantly increased, while their sensitivity to mechanical stress was enhanced in the diabetic mice. We found that cell proliferation was significantly decreased, while apoptotic cells were comparable in the skin of diabetic mice, compared to those in the control mice. In the epidermis, Keratin 5 and keratin 14 expressions were reduced, while keratin 10 and loricrin were ectopically induced in diabetic mice. These data suggest that hyperglycemia altered keratinocyte proliferation/differentiation. Finally, these phenotypes observed in diabetic mice were mitigated by insulin treatment. Reduction in basal cell number and perturbation of the proliferation/differentiation process could be the underlying mechanisms for impaired skin barrier functions in diabetic mice.

Epidermis-dermis junction as a novel location for bone marrow-derived cells to reside in response to ionizing radiation.

Bone marrow-derived cells (BMDCs) can migrate into the various organs in the mice irradiated by ionizing radiation (IR). However, it may not be the case in the skin. While IR is used for bone marrow (BM) transplantation, studying with the epidermal sheets demonstrated that the BMDC recruitment is extraordinarily rare in epidermis in the mouse. Herein, using the chimera mice with BM from green fluorescent protein (GFP) transgenic mice, we simply examined if BMDCs migrate into any layers in the total skin, as opposed to the epidermal sheets, in response to IR. Interestingly, we identified the presence of GFP-positive (GFP(+)) cells in the epidermis-dermis junction in the total skin sections although the epidermal cell sheets failed to have any GFP cells. To examine a possibility that the cells in the junction could be mechanically dissociated during separating epidermal sheets, we then salvaged such dissociated cells and examined its characteristics. Surprisingly, some GFP(+) cells were found in the salvaged cells, indicating that these cells could be derived from BM. In addition, such BMDCs were also associated with inflammation in the junction. In conclusion, BMDCs can migrate to and reside in the epidermis-dermis junction after IR.

Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA.

A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP) has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (-OH → -OTs (tosylate) → -N3) in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8) terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL) and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA) through electrostatic attraction, making them promising as nonviral vectors for gene delivery.

Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy.

Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin-treated mice, insulin- and TNF-α co-expressing bone marrow-derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high-fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co-expression of insulin and TNF-α in c-kit(+)Sca-1(+)lineage(-) (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.