Diabetic Microenvironment Research Articles

Dysregulation of Nrf2 redox pathway in macrophages under diabetic microenvironment.

In the present study, we characterized the aberration in Nrf2 signaling in macrophages under a hyperglycemic microenvironment that reflects diabetic wounds in vitro and studied the effect of an Nrf2 activator pterostilbene (PTS) in these experimental conditions. Macrophages were exposed to pro-inflammatory cytokines TNFα and IFNγ with (HG+) or without high glucose (NG+) followed by the treatment with or without PTS. Western blotting was undertaken to assess the Nrf2 translocation from cytosol to nucleus followed by its downstream and upstream mediators, heme oxygenase-1 and Akt, respectively, the latter via phosphorylation. Quantitative PCR was also carried out to check the expression of macrophage mannose receptor CD206. We found a 2-fold reduction in the activation of Nrf2 in the HG+ group at 24h compared to NG+, which was significantly improved by the treatment with PTS. Reduction in the levels of heme oxygenase-1 and phosphorylation of Akt in the HG+ group was also ameliorated by PTS. Furthermore, the gene expression of CD206 that was significantly reduced in the HG+ group was also restored by PTS treatment. The disruption of Nrf2 signaling in macrophages in a hyperglycemic microenvironment in vitro may indeed reflect diabetic wounds, as opposed to other non-diabetic wounds.

Exosomes: Biomarkers and Therapeutic Targets of Diabetic Vascular Complications

Diabetic vascular complications (DVC) including macrovascular and microvascular lesions, have a significant impact on public health, and lead to increased patient mortality. Disordered intercellular cascades play a vital role in diabetic systemic vasculopathy. Exosomes participate in the abnormal signal transduction of local vascular cells and mediate the transmission of metabolic disorder signal molecules in distant organs and cells through the blood circulation. They can store different signaling molecules in the membrane structure and release them into the blood, urine, and tears. In recent years, the carrier value and therapeutic effect of exosomes derived from stem cells have garnered attention. Exosomes are not only a promising biomarker but also a potential target and tool for the treatment of DVC. This review explored changes in the production process of exosomes in the diabetic microenvironment and exosomes’ early warning role in DVC from different systems and their pathological processes. On the basis of these findings, we discussed the future direction of exosomes in the treatment of DVC, and the current limitations of exosomes in DVC research.

Safeguarding Osteointegration in Diabetic Patients: A Potent “Chain Armor” Coating for Scavenging ROS and Macrophage Reprogramming in a Microenvironment‐Responsive Manner

AbstractRisk of implant failure increases profoundly in patients with pre‐existing conditions (e.g., diabetes). Current therapies adopt a one‐sided focus on the direct antibacterial properties of biomaterials and osteogenesis stimulation. However, in this study it is demonstrated that a “chain armor” structure (Ce‐TA) that mainly targets the regulation of the local pathological microenvironment, provides a novel solution to scavenge reactive oxygen species (ROS) by simulating superoxide dismutase and catalase and significantly improving osteointegration under diabetic conditions. Ce‐TA based on a metal phenolic network biological functional interface is successfully constructed. Ce‐TA, an ultrathin armor structure, is biocompatible and facile. Through in vitro assays it is demonstrated that Ce‐TA reshapes the diabetic microenvironment into a regenerative one in a microenvironment‐responsive manner, where Ce‐TA regulates hypoxia‐inducible factor 1α (HIF‐1α) activity by reducing the level of mitochondrial ROS, and effectively alleviates mitochondrial dysfunction and reprogrammes macrophages to a pro‐healing state. Furthermore, it is confirmed that Ce‐TA shows excellent therapeutic effects on the reducing postoperative infection and enhances osteointegration of intra‐osseous implants in diabetic rat models. The proposed strategy opens up a promising opportunity for repurposing metals with intrinsic enzyme‐like activity for the goal of enhancing the osteointegration of devices with orthopedic and dental applications among diabetic patients.

Breast milk MSCs upregulated β-cells PDX1, Ngn3, and PCNA expression via remodeling ER stress /inflammatory /apoptotic signaling pathways in type 1 diabetic rats

Type 1 diabetes mellitus (T1DM) is one of the autoimmune diseases characterized by beta-cell dysfunction with serious health complications. Br-MSCs represent a novel valid candidate in regenerative medicine disciplines. Yet, the full potential of Br-MSCs in managing type 1 diabetes remains elusive. Indeed, this study was designed to explore a novel approach investigating the possible regenerative capacity of Br-MSCs in type1 diabetic islet on the level of the cellular mRNA expression of different molecular pathways involved in pancreatic beta-cell dysfunction. Sixty adult male Sprague-Dawley rats were randomly assigned into 3 groups (20 rats each); the control group, type1 diabetic group, and the type 1 diabetic Br-MSCs treated group. And, for the first time, our results revealed that intraperitoneally transplanted Br-MSCs homed to the diabetic islet and improved fasting blood glucose, serum insulin level, pancreatic oxidative stress, upregulated pancreatic mRNA expression for: regenerative markers (Pdx1, Ngn3, PCNA), INS, beta-cell receptors (IRS1, IRβ, PPARγ), pancreatic growth factors (IGF-1, VEGFβ1, FGFβ), anti-inflammatory cytokine (IL10) and anti-apoptotic marker (BCL2) too, Br-MSCs downregulated pancreatic mRNA expression for: inflammatory markers (NFKβ, TNFα, IL1β, IL6, IL8, MCP1), apoptotic markers for both intrinsic and extrinsic pathways (FAS, FAS-L, P53, P38, BAX, Caspase3), ER stress markers (ATF6, ATF3, ATF4, BIP, CHOP, JNK, XBP1) and autophagy inhibitor (mTOR). In conclusion, Br-MSCs could be considered as a new insight in beta cell regenerative therapy improving the deteriorated diabetic islet microenvironment via modulating; ER stress, inflammatory, and apoptotic signaling pathways besides, switching on the cellular quality control system (autophagy) thus enhancing beta-cell function.

Impaired soft and hard callus formation during fracture healing in diet-induced obese mice as revealed by 3D contrast-enhanced computed tomography imaging.

The impact of diabetes mellitus on bone fracture healing is clinically relevant as the patients experience delayed fracture healing. Even though efforts have been made to understand the detrimental effects of type 2 diabetes mellitus (T2DM) on the fracture healing process, the exact mechanisms causing the pathophysiological outcomes remain unclear. The aim of this study was to assess alterations in bone fracture healing (tibial fracture surgery, intramedullary pinning) of diet-induced obese (DIO) mice, and to investigate the in vitro properties of osteochondroprogenitors derived from the diabetic micro-environment. High-resolution contrast-enhanced microfocus X-ray computed tomography (CE-CT) enabled a simultaneous 3D assessment of the amount and spatial distribution of the regenerated soft and hard tissues during fracture healing and revealed that osteogenesis as well as chondrogenesis are altered in DIO mice. Compared to age-matched lean controls, DIO mice presented a decreased bone volume fraction and increased callus volume and adiposity at day 14 post-fracture. Of note, bone turnover was found altered in DIO mice relative to controls, evidenced by decreased blood serum osteocalcin and increased serum CTX levels. The in vitro data revealed that not only the osteogenic and adipogenic differentiation of periosteum-derived cells (PDCs) were altered by hyperglycemic (HG) conditions, but also the chondrogenic differentiation. Elevated PPARγ expression in HG conditions confirmed the observed increase in differentiated adipocytes in vitro. Finally, chondrogenesis-related genes COL2 and COL10 were downregulated for PDCs treated with HG medium, confirming that chondrogenic differentiation is compromised in vitro and suggesting that this may affect callus formation and maturation during the fracture healing process in vivo. Altogether, these results provide novel insights into the alterations of long bone fracture repair and suggest a link between HG-induced dysfunctionality of osteochondroprogenitor differentiation and fracture healing impairment under T2DM conditions.

Pancreatic β-Cell Senescence: Mechanisms and Association with Diabetes

Senescence occurs as a part of the cellular response to different stressors. With increasing age, continuous exposure to stressors leads to age-induced senescence. Pancreatic β-cell proliferation and glucose homeostasis also decrease with age, which results in a decrease in β cell mass and, eventually, the possible development of diabetes. This process is mediated through impaired cell cycle regulators, along with specific increases in cell cycle inhibitors, telomere shortening, and defective DNA repair mechanisms. Diabetes contributes to β-cell senescence through hyperglycaemia, dyslipidaemia, oxidative stress, and inflammation. β cells isolated from patients with Type 2 diabetes mellitus have been shown to have senescence markers, such as senescence-associated secretory phenotype genes and β-galactosidase. In this paper, the authors discuss the mechanisms of cellular senescence, how senescence is impacted by the diabetic microenvironment, and the possible mechanisms and factors contributing to β-cell senescence.

High Glucose Restraint of Acetylcholine-Induced Keratinocyte Epithelial-Mesenchymal Transition Is Mitigated by p38 Inhibition

Non-neuronal acetylcholine (Ach) plays important roles in various aspects of cell biology and homeostasis outside the neural system. Keratinocytes (KCs) have a functional cholinergic mechanism, suggesting that they respond to Ach. However, the physiological role and mechanism by which Ach modulates wound KC behavior in both nondiabetic and diabetic conditions are unexplored. We found an enrichment in neurotransmitter-related pathways in microdissected-migrating nondiabetic and diabetic KCs. We showed that Ach upregulated TGFβRII through Src-extracellular signal‒regulated kinase 1/2 pathway to potentiate TGFβ1-mediated epithelial‒mesenchymal transition in normoglycemic condition. Unexpectedly, KCs were nonresponsive to the elevated endogenous Ach in a hyperglycemic environment. We further showed that the activation of p38 MAPK in high glucose condition interferes with Src-extracellular signal‒regulated kinase 1/2 signaling, resulting in Ach resistance that could be rescued by inhibiting p38 MAPK. A better understanding of the cholinergic physiology in diabetic KCs could improve wound management and care. The finding suggests that mitigating the inhibitory effect of diabetic wound microenvironment has a direct clinical implication on the efficacy and safety of various wound healing agents to improve chronic diabetic wounds.

Improving Chronic Diabetic Wound Healing through an Injectable and Self-Healing Hydrogel with Platelet-Rich Plasma Release.

Diabetic skin ulcer is one of the severe complications of diabetes mellitus, which has a high incidence and may cause death or disability. Platelet-rich plasma (PRP) is widely used in the treatment of diabetic wounds due to the effect of growth factors (GFs) derived from it. However, the relatively short half-life of GFs limits their applications in clinics. In addition, the presence of a large amount of proteases in the diabetic wound microenvironment results in the degradation of GFs, which further impedes angiogenesis and diabetic wound healing. In our study, we fabricated a self-healing and injectable hydrogel with a composite of chitosan, silk fibroin, and PRP (CBPGCTS-SF@PRP) for promoting diabetic wound healing. CBPGCTS-SF@PRP could protect PRP from enzymatic hydrolysis, release PRP sustainably, and enhance the chemotaxis of mesenchymal stem cells. The results showed that it could promote the proliferation of repair cells in vitro. Moreover, it could enhance wound healing by expediting collagen deposition, angiogenesis, and nerve repair in a type 2 diabetic rat model and a rat skin defect model. We hope that this study will offer a new treatment for diabetic nonhealing wounds in clinics.

The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes

BackgroundIn the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes.MethodsMouse GECs were exposed to high glucose media (HG) or 2.5% v/v serum from diabetic mice or serum from non-diabetic controls, and evaluated for mitochondrial function (oxygen consumption), structure (electron microscopy), morphology (mitotracker), mitochondrial superoxide (mitoSOX), as well as accumulation of oxidized products (DNA lesion frequency (8-oxoG, endo-G), double strand breaks (γ-H2AX), endothelial function (NOS activity), autophagy (LC3) and apoptotic cell death (Annexin/PI; caspase 3). Supernatant transfer experiments from GECs to podocytes were performed to establish the effects on podocyte survival and transwell experiments were performed to determine the effects in co-culture.ResultsDiabetic serum specifically causes mitochondrial dysfunction and mitochondrial superoxide release in GECs. There is a rapid oxidation of mitochondrial DNA and loss of mitochondrial biogenesis without cell death. Many of these effects are blocked by mitoTEMPO a selective mitochondrial anti-oxidant. Secreted factors from dysfunctional GECs were sufficient to cause podocyte apoptosis in supernatant transfer experiments, or in co-culture but this did not occur when GECs had been previously treated with mitoTEMPO.ConclusionDissecting the impact of the diabetic environment on individual cell-types from the kidney glomerulus indicates that GECs become dysfunctional and pathological to neighboring podocytes by increased levels of mitochondrial superoxide in GEC. These studies indicate that GEC-signaling to podocytes contributes to the loss of the glomerular filtration barrier in DKD.CcDz7iHMjDYmrcJBznsPoeVideo abstractGraphical abstract

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy.

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features.

Selenoprotein S attenuates endothelial dysfunction in a diabetic vascular chip.

Endothelial dysfunction (ED) is a critical and initiating factor in the genesis of diabetic vascular complications whose occurrence and development is closely related to the complex intravascular microenvironment. However, currently, there is no dynamic model simulating the diabetic vascular endothelial microenvironment that can be used to investigate the mechanism underlying multifactor-induced ED. Here, we developed an integrated microfluidic chip as a new methodological platform to study vascular ED. Selenoprotein S (SELENOS) was found to be involved in the defense against oxidative stress-induced vascular endothelial injury in our previous studies. However, the regulatory signaling pathway underlying this process has not been described. With this chip, we demonstrated that multifactor-induced oxidative stress injury in human aortic endothelial cells (HAECs) has a synergistic effects and upregulates SELENOS expression. Subsequently, SELENOS was found to protect HAECs against multifactor-induced oxidative stress injury by regulating the PKCα/PI3K/Akt/eNOS pathway in the diabetic vascular endothelial microenvironment. Based on these data, our diabetic vascular chip provides a promising tool for studying vascular endothelial function, and SELENOS may be a novel target for prevention and treatment of diabetic macrovascular complications.

Modulating neutrophil extracellular traps for wound healing.

A diabetic microenvironment primes neutrophils for NETosis, a process of formation of neutrophil extracellular traps (NETs) that further degrades the neutrophils and makes them unavailable for the early-stage inflammatory processes. Mechanistically, simple modification of arginine residues of histones to citrulline by peptidylarginine deiminase (PAD4) enzyme is considered to be a prerequisite for NETosis. In fact, under diabetic conditions, an increase in PAD4-mediated NET formation is considered as one of the reasons for impaired wound healing. Therefore, in the present work, an alginate-GelMa (generally recognized as safe category by FDA, USA) based hydrogel scaffold containing a tripeptide (Thr-Asp-F-amidine) that inhibits PAD4 is developed, based on the hypothesis that inhibiting PAD4 enzyme might offer a way to enhance wound healing under diabetic conditions. The scaffolds are thoroughly characterized for their physicochemical and biological properties. Furthermore, neutrophil-scaffold interactions in terms of NETosis ability and release of other related biomarkers are studied. The wound healing ability is evaluated by a cell migration assay. In vivo wound healing efficacy of the developed scaffolds is demonstrated using a diabetic rat model. The results suggest a reduction in NETosis in the presence of a PAD4 inhibitor. Thus, the study demonstrates a novel scaffold system to deliver the PAD4 inhibitor that can be used to modulate NETosis and improve wound healing.

Human mesenchymal stem cells-conditioned medium improves diabetic wound healing mainly through modulating fibroblast behaviors.

Fibroblast plays a key role in wound healing, and the advantages of mesenchymal stem cells (MSC) secretome in wound healing have previously been reported. In the present study, we investigated the impact of human bone marrow MSC-conditioned media (CM) on skin wound healing in diabetic rats and found that some improvements occurred mainly through fibroblast functions. Then, we scrutinized the impact of MSC-CM treatment on fibroblast cellular behavior by culturing human dermal fibroblasts (HDFs) in a high-glucose (HG) medium, as an in vitro diabetic model. In vivo findings revealed significant improvements in some healing kinetics of diabetic wound which received MSC-CM. Particularly, MSC-CM-treated diabetic wounds reached considerably higher percentages of wound closure. Also, the granulation tissue of these wound had less pronounced inflammatory response, better tissue remodeling, and more vascularization compared with non-treated diabetic ones. Gene expression analyses indicated that MSC-CM treatment leads to upregulation of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) genes. In addition, a significantly higher cell viability/proliferation, migration, and bFGF gene expression were observed when MSC-CM was used to treat HDFs in HG culture media. Based on these findings, it is suggested that MSC-CM could promote wound repair and skin regeneration, in some major processes, via improvement of cellular behaviors of fibroblasts in the diabetic microenvironment. The beneficial advantages of mesenchymal stem cells-conditioned media on fibroblast cellular behaviors and wound healing may lead to establish a novel approach as an alternative therapeutic procedure to cure chronic wounds in diabetic conditions.

Mechanism and Consequences of The Impaired Hif-1\u03b1 Response to Hypoxia in Human Proximal Tubular HK-2 Cells Exposed to High Glucose

Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.

MiRNAs responsive to the diabetic microenvironment in the human beta cell line EndoC-βH1 may target genes in the FOXO, HIPPO and Lysine degradation pathways

Altered expression of miRNAs is evident in the islets of diabetic human donors, but the effects of specific aspects of the diabetic microenvironment and identity of gene ontology pathways demonstrating target gene enrichment in response to each is understudied.We assessed changes in the miRNA milieu in response to high/low glucose, hypoxia, dyslipidaemia and inflammatory factors in a humanised EndoC-βH1 beta cell culture system and performed miRPath analysis for each treatment individually. The 10 miRNAs demonstrating the greatest dysregulation across treatments were then independently validated and Gene Set Enrichment Analysis to confirm targeted pathways undertaken.171 of 392 miRNAs displayed altered expression in response to one or more cellular stressors. miRNA changes were treatment specific, but their target genes were enriched in conserved pathways. 5 miRNAs (miR-136-5p, miR299-5p, miR-454-5p, miR-152 and miR-185) were dysregulated in response to multiple stressors and survived validation in independent samples (p = 0.008, 0.002, 0.012, 0.005 and 0.024 respectively). Target genes of dysregulated miRNAs were clustered into FOXO1, HIPPO and Lysine degradation pathways (p = 0.02, p = 5.84 × 10−5 and p = 3.00 × 10−3 respectively).We provide evidence that the diabetic microenvironment may induce changes to the expression of miRNAs targeting genes enriched in pathways involved in cell stress response and cell survival.

2219-PUB: Antioxidant Protective Effect and Mechanism of Selenoprotein S in Diabetic Vascular Endothelium-Mimicking Microenvironment

Endothelial dysfunction is an initiating key step for diabetic macrovascular complications, and it is closely related to the hemodynamic microenvironment in vivo. However, diabetic patients usually coexist with other cardiovascular risk factors, and the more powerful targeting strategy for protecting endothelium is still limited. Selenoprotein S (SelS) was found in our previous study that could increase vascular endothelial cells resistance to oxidative stress associated with protein kinase C α (PKCα). Thus, in this study, we constructed a diabetic vascular endothelial bionic microenvironment mimicking hyperglycemia and/or hyperlipidemia based on microfludic chip through modulating the concentration of medium glucose and oxidized low density lipoproteins. Our results showed that under diabetic vascular endothelial bionic microenvironment, the upregulation of SelS enhanced nitric oxide synthase (eNOS) expression and reduced endothelin-1 level in human aortic endothelial cells (HAECs), accompany with increase in cell viability and SOD1 and SOD2 activity, and decrease in ROS. Furthermore, SelS overexpression attenuated the activation of PKCα, which subsequently promoted the activation of PI3K/Akt/eNOS pathway. Conversely, the knockdown of SelS resulted in an enhancement of endothelial injury. These findings suggest that SelS could protect endothelial cells from oxidative stress injury under diabetic vascular endothelial bionic microenvironment through PKCα and PI3K/Akt/eNOS pathway. Our results offer a promising and biomimetic platform for investigation on endothelial protection, and provide evidence of SelS as potential novel target to treat diabetic macrovascular complications. Disclosure S. Yu: None. Y. Zhong: None. L. Men: None. J. Yao: None. J. Du: None. Funding National Natural Science Foundation of China (81570727)

Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.

Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.

Overexpression of Sirt6 promotes M2macrophage transformation, alleviating renal injury in diabetic nephropathy.

In this study, we aimed to investigate the associations between Sirt6, macrophages and diabetic nephropathy (DN). Immunohistochemical, western blot and RT-qPCR analyses were performed to detect the expression levels of Sirt6, the markers of podocytes and monocytes and related inflammatory factors in the tissues of rats with streptozocin-induced DN. A series of cell experiments in isolated culture or the co-culture of macrophages and podocytes were conducted to examine the effects of the overexpression of Sirt6 on macrophage transformation, podocyte apoptosis and associated genes, and analyses were performed using RT-qPCR, flow cytometry and western blot analysis, where appropriate. In the rat model of DN, injured podocytes were represented by the decreased protein expression levels of Nephrin and Sirt6, and by an increased Desmin expression. Additionally, the M1 phenotype transformation of macrophages was evidenced by the increased expression levels of CD86, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), and by the decreased expression levels of CD206, Sirt6, interleukin (IL)-4 and IL-10. In vitro assays of macrophages and podocytes demonstrated that glucose promoted macrophage M1 transformation and podocyte apoptosis in a dose-dependent manner and attenuated Sirt6 expression. Macrophages transformed into the M2 phenotype following the overexpression of Sirt6 by the successful transfection of macrophages with a Sirt6 overexpression plasmid. Sirt6 was also overexpressed in podocytes. In a Transwell co-culture system, the overexpression of Sirt6 in macrophages (but not the overexpression of Sirt6 in podocytes) protected the podocytes from high-glucose-induced injury. However, the apoptosis of the podocytes overexpressing Sirt6 (induced by transfection with a Sirt6 overexpression plasmid) still increased when these podocytes were co-cultured with macrophages in high-glucose medium. These protective effects were evidenced by the inhibition of apoptosis, the upregulation of the expression levels of Bcl-2 and CD206, as well as by the decreased expression levels of Bax and CD86. On the whole, the findings of this study suggest that Sirt6 protects podocytes against injury in a mimicked diabetic kidney microenvironment by activating M2 macrophages, indicating that Sirt6 can act as an immune response regulatory factor in DN-associated renal inflammatory injury.

Long-Term Diabetic Microenvironment Augments the Decay Rate of Capsaicin-Induced Currents in Mouse Dorsal Root Ganglion Neurons.

Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.

Promoting Angiogenesis in Oxidative Diabetic Wound Microenvironment Using a Nanozyme-Reinforced Self-Protecting Hydrogel.

Impaired diabetic wound healing represents a devastating and rapidly growing clinical problem associated with high morbidity, mortality, and recurrence rates. Engineering therapeutic angiogenesis in the wounded tissue is critical for successful wound healing. However, stimulating functional angiogenesis of the diabetic wound remains a great challenge, due to the oxidative damage and denaturation of bio-macromolecule-based angiogenic agents in the oxidative diabetic wound microenvironment. Here, we present a unique “seed-and-soil” strategy that circumvents the limitation by simultaneously reshaping the oxidative wound microenvironment into a proregenerative one (the “soil”) and providing proangiogenic miRNA cues (the “seed”) using an miRNA-impregnated, redox-modulatory ceria nanozyme-reinforced self-protecting hydrogel (PCN-miR/Col). The PCN-miR/Col not only reshapes the hostile oxidative wound microenvironment, but also ensures the structural integrity of the encapsulated proangiogenic miRNA in the oxidative microenvironment. Diabetic wounds treated with the PCN-miR/Col demonstrate a remarkably accelerated wound closure and enhanced quality of the healed wound as featured by highly ordered alignment of collagen fiber, skin appendage morphogenesis, functional new blood vessel growth, and oxygen saturation.