Diabetic Murine Model Research Articles

Observation of neutrophil extracellular traps in the development of diabetic nephropathy using diabetic murine models.

Diabetic nephropathy (DN) is a progressive complication among patients with diabetes and the most common cause of end-stage kidney disease. Neutrophil extracellular traps (NETs) are known to play a role in kidney disease, thus this study aimed to determine their role in the development of diabetic kidney disease using diabetic murine models. Protein and histological analyses revealed that db/db mice and streptozotocin DN models expressed no significant NET-related proteins, myeloperoxidase, citrullinated histone H3 (citH3), neutrophil elastase, and lymphocyte antigen 6 complex locus G6D (Ly6G). However, the inflamed individuals in the DN model showed that citH3 and Ly6G were highly deposited in the renal system based on immunohistochemistry images. In vitro, NET treatment did not induce apoptosis in glomerular endothelial and renal tubular epithelial cells. NET inhibition by DNase administration demonstrated no significant changes in cell apoptosis. NET-related proteins were only expressed in the DN model with tubulointerstitial inflammation. Our study revealed that NETs are only induced in mice with hyperglycemia-induced inflammation.

Downregulation of Neuronal Nitric Oxide Synthase (nNOS) within the Paraventricular Nucleus in Ins2Akita-type-1 Diabetic Mice Contributes to Sympatho-excitation

Activation of both renin-angiotensin system (RAS) and the sympathetic system is the primary etiologic event in developing cardiovascular complications in diabetes mellitus (DM). However, the precise mechanisms for sympathetic activation in DM have not been elucidated. Here we attempted to investigate diabetes-linked cardiovascular dysregulation due to angiotensin II (Ang II)-mediated reduction in neuronal nitric oxide (NO) synthase (nNOS) within the paraventricular neuleus (PVN). In the present study, we used Ins2+/-Akita (a spontaneous, insulin-dependent genetic diabetic non-obese murine model) and wild-type (WT) littermates mice as controls. At 14 weeks of age, we found the Akita mice had increased renal sympathetic nerve activity and elevated levels of plasma norepinephrine. There was decreased expression of nNOS protein (Akita 0.43 ± 0.11 vs. WT 0.75 ± 0.05, P < 0.05) in the PVN of Akita mice. Akita mice had increased expression of angiotensin-converting enzyme (ACE) (Akita 0.58 ± 0.05 vs. WT 0.34 ± 0.04, P < 0.05) and Ang II type 1 receptor (Akita 0.49 ± 0.03 vs. WT 0.29 ± 0.09, P < 0.05), decreased expressions of ACE2 (Akita 0.17 ± 0.05 vs. WT 0.27 ± 0.03, P < 0.05) and angiotensin (1-7) Mas receptor (Akita 0.46 ± 0.02 vs. WT 0.77 ± 0.07, P < 0.05). Futher, there were increased protein levels of protein inhibitor of nNOS (PIN) (Akita 1.75 ± 0.08 vs. WT 0.71 ± 0.09, P < 0.05) with concomitantly decreased catalytically active dimers of nNOS (Akita 0.11 ± 0.04 vs. WT 0.19 ± 0.02, P < 0.05) in the PVN in Akita mice. Our studies suggest that activation of the excitatory arm of RAS, leads to a decrease NO, causing an over-activation of the sympathetic drive in DM.

Chronic hyperglycemia alters retinal astrocyte microstructure and uptake of cholera toxin B in a murine model of diabetes.

Astrocytes are the principle glial cells of the central nervous system and play an active role in maintaining proper metabolism in surrounding neurons. Because of their involvement in metabolic control, it is likely that their physiology changes in response to metabolic diseases such as diabetes and associated diabetic retinopathy. Here, we investigated whether microstructural changes in astrocyte morphology occur during the early stages of chronic hyperglycemia that may be indicative of early pathogenic programs. We used MORF3 mice in conjunction with streptozotocin-induced hyperglycemia to investigate the morphology of single retinal astrocytes at an early timepoint in diabetic disease. We report that astrocytes initiate a morphological remodeling program, which depends on both the glycemic background and the presence of intravitreal injury, to alter the amount of the neuronal-associated pad and bristle microstructural motifs. Additionally, hyperglycemia increases astrocyte uptake of cholera toxin B, possibly reflecting changes in glycolipid and glycoprotein biosynthesis. Chronic hyperglycemia coupled with intravitreal injection of cholera toxin B also causes extensive leukocyte infiltration into the retina. Our results have important clinical relevance as current therapies for diabetic retinopathy involve intravitreal injection of pharmaceuticals in individuals with often poorly controlled blood glucose levels.

Evaluation of a mucoadhesive auto-nanoemulsifying drug delivery system (SNEDDS) for oral insulin administration

This study investigated the potential of self-nanoemulsifying drug delivery systems (SNEDDS) to optimize the oral bioavailability of insulin. Insulin complexes with phospholipids and enzymatically-modified phospholipids were developed and incorporated into the SNEDDS using Lauroglycol FCC as the oily phase and Cremophor EL and Labrafil M1944CS as the surfactant and co-surfactant, respectively. Additionally, mucoadhesive polysaccharides (sodium alginate and guar gum) were added further to enhance the bioavailability of insulin in these systems. The objective was to increase the bioavailability and bioactivity of an insulin-modified phosphatidylcholine complex by incorporating mucoadhesives into the SNEDDS. After polymer inclusion, the resulting nanoemulsions exhibited droplet diameters ranging from 57 to 83 nm. Cytotoxicity and apparent permeability tests were conducted on Caco-2 and NIH 3 T3 cell lines, revealing that toxicity was related to the concentrations of insulin and surfactant in the nanosystems—formulations containing guar gum as a mucoadhesive showed better tolerance to cell death in the Caco-2 line. In a murine diabetes model, the SNEDDS were observed to reduce glucose levels by up to 61.63 %, with a relative bioavailability of 2.25 % compared to subcutaneously administered insulin. These results suggest that SNEDDS incorporating mucoadhesives could represent a promising strategy for improving oral insulin delivery.

Global genomic profile of hippocampal endothelial cells by single-nuclei RNA sequencing in female diabetic mice is associated with cognitive dysfunction.

Type II diabetes mellitus (T2D) is a chronic metabolic disease and a risk factor for cardiovascular disease and cerebrovascular dysfunction including vascular dementia. Sex differences in the prevalence of T2D, dementia, and global genomic changes in the brain have been observed; however, most studies have been performed in males. Therefore, our aim was to evaluate the consequence of T2D on cognitive function and decipher the underlying molecular transcriptomic mechanisms of endothelial cells in an important brain memory center, the hippocampus, using a female murine diabetes model. We assessed cognitive function, metabolic parameters, and then performed hippocampal single-nuclei RNA sequencing (snRNA seq) in adult female db/db and control wild-type (WT) mice. db/db mice exhibited characteristic T2D metabolism with hyperglycemia, hyperinsulinemia, and hyperlipidemia when compared with WT mice. Female db/db mice presented cognitive decline compared with wild-type mice, as determined by open field and Morris water maze tests. snRNAseq showed that T2D induced significant changes in the global transcriptomic profile of hippocampal endothelial cells by modulating the expression of not only protein-coding genes but also long noncoding RNAs. These genes regulate cell-cell junctions, cell chemotaxis, actin cytoskeleton organization, and cell adhesion, suggesting that diabetes increases endothelial cell permeability. Observed genomic changes also correlated with the genetics of persons with clinical Alzheimer's disease and vascular dementia. In conclusion, T2D, by transcriptional and posttranscriptional regulation, regulates endothelial cell dysfunction predictive of increased vascular permeability, and negatively impacts cognitive function. Our work has implications for sex-specific molecular therapeutic targets for dementia in females.NEW & NOTEWORTHY Female db/db mice presented cognitive decline as determined by open field and Morris water maze tests. snRNAseq showed that T2D induced changes in the global transcriptomic profile of hippocampal endothelial cells by modulating the expression of not only protein-coding genes but also long noncoding RNAs. These genes regulate cell-cell junctions, cell chemotaxis, or cell adhesion, suggesting increased endothelial permeability. Genomic changes correlated with the genetics of persons with clinical Alzheimer's disease and vascular dementia.

Immunomodulatory hydrogel orchestrates pro-regenerative response of macrophages and angiogenesis for chronic wound healing

Chronic wound healing often encounters challenges characterized by prolonged inflammation and impaired angiogenesis. While the immune response plays a pivotal role in orchestrating the intricate process of wound healing, excessive inflammation can hinder tissue repair. In this study, a bilayer alginate hydrogel system encapsulating polyelectrolyte complex nanoparticles (PCNs) loaded with anti-inflammatory cytokines and angiogenic growth factors is developed to address the challenges of chronic wound healing. The alginate hydrogel is designed using two distinct crosslinking methods to achieve differential degradation, thereby enabling precise spatial and temporal controlled release of PCNs. Initially, interleukin-10 (IL-10) is released to mitigate inflammation, while unsaturated PCNs bind and remove accumulated pro-inflammatory cytokines at the wound site. Subsequently, angiogenic growth factors, including vascular endothelial growth factor and platelet-derived growth factor, are released to promote vascularization and vessel maturation. Our results demonstrate that the bilayer hydrogel exhibits distinct degradation kinetics between the two layers, facilitating the staged release of multiple signaling molecules. In vitro experiments reveal that IL-10 can activate the Jak1/STAT3 pathway, thereby suppressing pro-inflammatory cytokines and chemokines while down-regulating inflammation-related genes. In vivo studies demonstrate that application of the hydrogel in chronic wounds using diabetic murine model promotes healing by positively modulating multiple integral reparative mechanisms. These include reducing inflammation, promoting macrophage polarization towards a pro-regenerative phenotype, enhancing keratinocyte migration, stimulating angiogenesis, and expediting wound closure. In conclusion, our hydrogel system effectively mitigates inflammatory responses and provides essential physiological cues by inducing a synergistic angiogenic effect, thus offering a promising approach for the treatment of chronic wounds.

Reduction of hyperglycemia in STZ-induced diabetic mice by prophylactic treatment with heat-killed Mycobacterium aurum: possible effects on glucose utilization, mitochondrial uncoupling, and oxidative stress in liver and skeletal muscle.

In addition to conventional treatment and modifications in physical activity and diet, alternative strategies have been investigated to manage, prevent, or delay diabetes in humans. In this regard, one strategy has relied on the immunomodulatory properties of mycobacteria, whereby Bacillus Calmette-Guerin, an attenuated live strain of Mycobacterium bovis, has been shown to improve glycemic control in patients with diabetes and to alleviate hyperglycemia in selected murine models of diabetes. A novel heat-killed (HK) whole-cell preparation of Mycobacterium aurum (M. aurum) is currently under development as a potential food supplement; nevertheless, its potential bioactivity remains largely unknown. Thus, the present study investigated the potential prophylactic anti-diabetic effects of HK M. aurum in streptozotocin (STZ)-induced diabetic mice. Mice were divided into three groups: the STZ-induced diabetic group was injected with a single intraperitoneal high dose of STZ, the HK M. aurum-treated diabetic group was prophylactically treated with three doses of HK M. aurum 6 weeks before STZ injection, and the control non-diabetic group was given three intradermal injections of borate-buffered saline and an intraperitoneal injection of citrate buffer. Liver lactate dehydrogenase (LDH), uncoupling protein 2 (UCP2), and glucose transporter 2 (GLUT2) and skeletal muscle LDH, UCP3, and GLUT4 protein expression levels in different mouse groups were determined by Western blot. Our results indicated that HK M. aurum did not cause any significant changes in glycemic levels of normal non-diabetic mice. Prophylactic administration of three doses of HK M. aurum to diabetic mice resulted in a significant reduction in their blood glucose levels when compared to those in control diabetic mice. Prophylactic treatment of diabetic mice with HK M. aurum significantly restored their disturbed protein expression levels of liver UCP2 and LDH as well as of skeletal muscle UCP3. On the other hand, prophylactic treatment of diabetic mice with HK M. aurum had no significant effect on their liver GLUT2 and skeletal muscle GLUT4 and LDH protein expression levels. Our findings provide the first evidence that HK M. aurum possesses a hyperglycemia-lowering capacity and might support its future use as a food supplement for the amelioration of diabetes.

Cardiac Troponin to Adjudicate Subclinical Heart Failure in Diabetic Patients and a Murine Model of Metabolic Syndrome.

Cardiovascular disease, kidney health, and metabolic disease (CKM) syndrome is associated with significant morbidity and mortality, particularly from congestive heart failure (CHF). Guidelines recommend measurement of cardiac troponin (cTn) to identify subclinical heart failure (HF) in diabetics/CKM. However, appropriate thresholds and the impact from routine screening have not been elucidated. cTnI was assessed using the Abbott high sensitivity (hs)-cTnI assay in outpatients with physician-ordered hemoglobin A1c (Hb A1c) and associated with cardiac comorbidities/diagnoses, demographics, and estimated glomerular filtration rate (eGFR). Risk thresholds used in CKM staging guidelines of >10 and >12 ng/L for females and males, respectively, were used. Multivariate logistic regression was applied. hs-cTnI was assessed in a high-fat-diet induced murine model of obesity and diabetes. Of 1304 patients, 8.0% females and 15.7% males had cTnI concentrations above the risk thresholds. Thirty-one (4.2%) females and 23 (4.1%) males had cTnI above the sex-specific 99% upper reference limit. A correlation between hs-cTnI and Hb A1c (R = 0.2) and eGFR (R = -0.5) was observed. hs-cTnI concentrations increased stepwise based on A1C of <5.7% (median = 1.5, IQR:1.3-1.8), 5.7%-6.4% (2.1, 2.0-2.4), 6.5%-8.0% (2.8, 2.5-3.2), and >8% (2.8, 2.2-4.3). Male sex (P < 0.001), eGFR (P < 0.001), and CHF (P = 0.004) predicted elevated hs-cTnI. Obese and diabetic mice had increased hs-cTnI (7.3 ng/L, 4.2-10.4) relative to chow-fed mice (2.6 ng/L, 1.3-3.8). A high proportion of outpatients with diabetes meet criteria for subclinical HF using hs-cTnI measurements. Glucose control is independently associated with elevated cTnI, a finding replicated in a murine model of metabolic syndrome.

OxInflammation Affects Transdifferentiation to Myofibroblasts, Prolonging Wound Healing in Diabetes: A Systematic Review.

Skin wounds, primarily in association with type I diabetes mellitus, are a public health problem generating significant health impacts. Therefore, identifying the main pathways/mechanisms involved in differentiating fibroblasts into myofibroblasts is fundamental to guide research into effective treatments. Adopting the PRISMA guidelines, this study aimed to verify the main pathways/mechanisms using diabetic murine models and analyze the advances and limitations of this area. The Medline (PubMed), Scopus, and Web of Science platforms were used for the search. The studies included were limited to those that used diabetic murine models with excisional wounds. Bias analysis and methodological quality assessments were undertaken using the SYRCLE bias risk tool. Eighteen studies were selected. The systematic review results confirm that diabetes impairs the transformation of fibroblasts into myofibroblasts by affecting the expression of several growth factors, most notably transforming growth factor beta (TGF-beta) and NLRP3. Diabetes also compromises pathways such as the SMAD, c-Jun N-terminal kinase, protein kinase C, and nuclear factor kappa beta activating caspase pathways, leading to cell death. Furthermore, diabetes renders the wound environment highly pro-oxidant and inflammatory, which is known as OxInflammation. As a consequence of this OxInflammation, delays in the collagenization process occur. The protocol details for this systematic review were registered with PROSPERO: CRD42021267776.

Diverse calcium signaling profiles regulate migratory behavior in avascular wound healing and aberrant signal hierarchy occurs early in diabetes.

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

The Ethyl Acetate Extract of Phyllanthus emblica L. Alleviates Diabetic Nephropathy in a Murine Model of Diabetes.

Oil-Gan is the fruit of the genus Phyllanthus emblica L. The fruits have excellent effects on health care and development values. There are many methods for the management of diabetic nephropathy (DN). However, there is a lack of effective drugs for treating DN throughout the disease course. The primary aim of this study was to examine the protective effects (including analyses of urine and blood, and inflammatory cytokine levels) and mechanisms of the ethyl acetate extract of P. emblica (EPE) on db/db mice, an animal model of diabetic nephropathy; the secondary aim was to examine the expression levels of p- protein kinase Cα (PKCα)/t-PKCα in the kidney and its downregulation of vascular endothelial growth factor (VEGF) and fibrosis gene transforming growth factor-β1 (TGF-β1) by Western blot analyses. Eight db/m mice were used as the control group. Forty db/db mice were randomly divided into five groups. Treatments included a vehicle, EPE1, EPE2, EPE3 (at doses of 100, 200, or 400 mg/kg EPE), or the comparative drug aminoguanidine for 8 weeks. After 8 weeks of treatment, the administration of EPE to db/db mice effectively controlled hyperglycemia and hyperinsulinemia by markedly lowering blood glucose, insulin, and glycosylated HbA1c levels. The administration of EPE to db/db mice decreased the levels of BUN and creatinine both in blood and urine and reduced urinary albumin excretion and the albumin creatine ratio (UACR) in urine. Moreover, EPE treatment decreased the blood levels of inflammatory cytokines, including kidney injury molecule-1 (KIM-1), C-reactive protein (CRP), and NLR family pyrin domain containing 3 (NLRP3). Our findings showed that EPE not only had antihyperglycemic effects but also improved renal function in db/db mice. A histological examination of the kidney by immunohistochemistry indicated that EPE can improve kidney function by ameliorating glomerular morphological damage following glomerular injury; alleviating proteinuria by upregulating the expression of nephrin, a biomarker of early glomerular damage; and inhibiting glomerular expansion and tubular fibrosis. Moreover, the administration of EPE to db/db mice increased the expression levels of p- PKCα/t-PKCα but decreased the expression levels of VEGF and renal fibrosis biomarkers (TGF-β1, collagen IV, p-Smad2, p-Smad3, and Smad4), as shown by Western blot analyses. These results implied that EPE as a supplement has a protective effect against renal dysfunction through the amelioration of insulin resistance as well as the suppression of nephritis and fibrosis in a DN model.

Co-Delivery of Recombinant Super-Affinity VEGF and PDGF-BB Accelerates Diabetic Wound Healing by Improving Angiogenesis, Arteriogenesis and Blood Flow

Abstract Background Diabetic ulcers are characterized by local ischemia, usually without macroangiopathy. We have previously found that the combination of VEGF and PDGF-BB in a fibrin matrix is effective in stimulating arterial growth to diabetic wounds. Aims We developed modified forms of VEGF and PDGF-BB to bear super-affinity for the extracellular matrix (SA-VP), in order to decorate the wound tissue without the need for a biomaterial. We investigated the dose-dependent effects on local blood flow and wound healing, by applying the treatment either to the healthy tissue around the wound or to the damaged wound bed. Methods Full thickness wounds were created on the dorsal skin of diabetic (db/db) mice. SA-VP or saline were applied to the wound bed or divided into 4 injections around the wound edge. Wound closure and blood flow were measured on days 4 and 8 after treatment and tissues were harvested for histology on day 8. Data were analysed by Anova with Tukey’s post-hoc test for multiple comparisons. Results Wound healing was improved dose-dependently by SA-VP by both delivery routes. Histologically, angiogenic areas and total amount of angiogenesis were significantly increased by the highest SA-VP dose, again independent of delivery route (p = 0.034). However, the total amount of recruited arterioles was significantly increased only in the healthy tissue around the wound and only by intradermal injections of SA-VP (p = 0.008). In agreement with the arteriogenic effect, the blood flow at day 4 was significantly increased only by injections of the highest SA-VP dose around the wound (p = 0.018). Conclusion SA-VP treatment significantly accelerates wound healing in a diabetic murine model with a clear dose-dependent effect. Targeting the undamaged tissue around the wound by injections induces more effective arteriogenesis. However, direct treatment of the wound bed is equally effective for healing. These results support further development for a potential clinical use.

Abstract 1101: Tnfa-setdb2 Pathway Regulates Fibroblast To Myofibroblast Transition In Wound Repair

Effective healing following tissue injury is predicated on an intimate coordination between immune cells and structural cells, where recruited immune cells induce or repress structural cell proliferation and gene expression. Early after tissue injury, fibroblasts begin to transition to myofibroblasts to contract and close wounds. In pathologic conditions where wounds fail to heal (such as diabetes), fibroblasts do not increase myofibroblast gene expression; however the mechanisms that control this fibroblast transition, in normal or diabetic conditions are not defined. Our group has previously identified that epigenetic alterations can control these phenotypic switches in immune and structural cells. Here, using human wound scSeq and murine transgenic models, we examined the mechanisms that may regulate this transition in the setting of injury. First, we identified that dermal fibroblasts when treated with the pro-inflammatory cytokine TNFa, significantly repressed myofibroblast genes including acta2, tgln, myl9, cald1, fibronectin and periostin. To identify possible the epigenetic mechanisms downstream of TNFa that modulate fibroblast phenotype, we performed an unbiased epigenetic superarray on TNFa-stimulated dermal fibroblasts, where SETDB2 was significantly upregulated and was also confirmed by qPCR. SiRNA-mediated knockdown of Setdb2 in murine fibroblasts prevented TNFa-mediated repression of myofibroblast genes, suggesting regulation of this transition by Setdb2. Human scSeq identified increased Setdb2 associated with increased TNFa signaling in wound fibroblasts, suggesting an interaction of these pathways. Interestingly, we found increased TNFa in macrophages and SETDB2 in fibroblasts from human diabetic wound scSeq and a murine diabetic (DIO) model, suggesting this pathway may contribute to the decreased conversion of fibroblasts to myofibroblasts seen in non-healing diabetic wounds. This work identifies setdb2 as a TNFa-mediated regulator of fibroblast to myofibroblast transition in wound repair and may identify a new therapeutic target for impaired tissue regeneration.

Abstract 3031: Novel Therapeutic Approach In Diabetic Wound Healing Using Inducible Mesenchymal Stem Cell-derived Fibroblasts In A Unique Murine Model

Introduction: It is estimated that about 40 to 60 million people in the United States suffer from diabetic foot ulcers (DFUs), one of the most severe DM complications. A significant proportion of affected individuals undergo amputation and are exposed to high mortality risks. Stem cell-based therapies offer a promising therapeutic avenue for these patients. Fibroblasts hold a crucial role in wound healing and angiogenesis, and we further highlighted that activated Notch signaling in fibroblasts derived from diabetic wounds leads to dysregulated fibroblast function. In this study, the potential therapeutic outcomes from genetically modified fibroblasts derived from mesenchymal stem cells (MSC-DF) in treating DFU is assessed, utilizing a unique genetic mouse model. Methods: A fibroblast-specific and inducible diabetic murine model with triple-gene engineering was created via inducible intracellular Notch1 pathway activation (RosaN1IC/Col1a2-Cre/ERT/Leprdb). MSC-DFs harvested from 10-12 week old B6db/ ROSAN1I/ / Cola2-Cre/ERT (LLRRCc) donor mice were cultured in vitro with activation versus silencing of Notch 1 signaling. Recipient LLRRCc mice (8-12-week-old) underwent a 6mm excisional dorsal skin wound and subsequent local injection of 1x10^6 donor syngeneic activated or silenced GOF Notch1 -MSC-DFs (n= 5/group). Wound closure was monitored daily for 9 consecutive days post-injury, and tissue was obtained for histological sectioning. Results: GOF Notch1 -MSC-DFs were characterized by immunostaining based on expression of markers specific to fibroblasts and a panel of Notch signaling genes. LLRRCc murine subjects receiving silenced GOF Notch1 -MSC-DFs treatment (n=5) demonstrated faster rates of wound closure over activated GOF Notch1 -MSC-DFs (n=5) treated mice. Collagen deposition was more abundant in wounds treated with silenced GOF Notch1 -MSC-DFs. Conclusion: MSC-DF deactivated Notch1 signaling therapy improves the rate of wound healing in GOF Notch Diabetic mice. These promising outcomes demonstrate a potential role for this innovative stem cell-based therapy targeting Notch-1-modified MSC-DF in future clinical applications for DFU patients through restoration of impaired fibroblast function.

Extracellular metallothionein as a therapeutic target in the early progression of type 1 diabetes.

Type 1 diabetes (T1D) is characterized by lymphocyte infiltration into the pancreatic islets of Langerhans, leading to the destruction of insulin-producing beta cells and uncontrolled hyperglycemia. In the nonobese diabetic (NOD) murine model of T1D, the onset of this infiltration starts several weeks before glucose dysregulation and overt diabetes. Recruitment of immune cells to the islets is mediated by several chemotactic cytokines, including CXCL10, while other cytokines, including SDF-1α, can confer protective effects. Global gene expression studies of the pancreas from prediabetic NOD mice and single-cell sequence analysis of human islets from prediabetic, autoantibody-positive patients showed an increased expression of metallothionein (MT), a small molecular weight, cysteine-rich metal-binding stress response protein. We have shown that beta cells can release MT into the extracellular environment, which can subsequently enhance the chemotactic response of Th1 cells to CXCL10 and interfere with the chemotactic response of Th2 cells to SDF-1α. These effects can be blocked in vitro with a monoclonal anti-MT antibody, clone UC1MT. When administered to NOD mice before the onset of diabetes, UC1MT significantly reduces the development of T1D. Manipulation of extracellular MT may be an important approach to preserving beta cell function and preventing the development of T1D.

Optimization of ultrasound contrast agent and treatment duration for drug delivery to methicillin-resistant Staphylococcus aureus diabetic wound biofilms in mice

Chronic wounds are frequently infected with bacterial biofilms which pose a barrier to drug diffusion, drug uptake, and facilitate drug tolerance, thereby prolonging the healing process in chronic wounds and increasing the likelihood of relapse. A major contributor to relapse is persister cells, a subset resilient to high antibiotic doses. Previous work by our group showcased significantly improved gentamicin efficacy in a diabetic murine wound model using ultrasound, nanodroplets, and an anti-persister drug. Here, we aim to optimize nanodroplet formulation for persister cell targeting and treatment duration for clinical translation. In a methicillin-resistant Staphylococcus aureus (MRSA) diabetic wound model in SKH-1 hairless mice (n = 5), wounds were treated twice daily with gentamicin, ultrasound, and nanodroplets containing oxygen or palmitoleic acid (PA). These components aim to increase oxygenation and enhance persister uptake, respectively. Additionally, treatments with gentamicin, PA nanodroplets, and ultrasound for 2, 5, and 10 minutes were investigated (n = 3). Preliminary findings indicate PA most effectively potentiates antibiotic activity, 10- minute treatments improve efficacy by 1-log compared to 5-minutes, and 2-minute treatments achieve comparable efficacy to 5-minutes. These initial findings suggest a promising strategy for enhancing antibiotic efficacy and targeting persister cells in biofilms, with consideration for resource-efficient treatment times for clinical application.

Hydrogel dressings with intrinsic antibiofilm and antioxidative dual functionalities accelerate infected diabetic wound healing

Chronic wounds are often infected with biofilm bacteria and characterized by high oxidative stress. Current dressings that promote chronic wound healing either require additional processes such as photothermal irradiation or leave behind gross amounts of undesirable residues. We report a dual-functionality hydrogel dressing with intrinsic antibiofilm and antioxidative properties that are synergistic and low-leaching. The hydrogel is a crosslinked network with tethered antibacterial cationic polyimidazolium and antioxidative N-acetylcysteine. In a murine diabetic wound model, the hydrogel accelerates the closure of wounds infected with methicillin-resistant Staphylococcus aureus or carbapenem-resistant Pseudomonas aeruginosa biofilm. Furthermore, a three-dimensional ex vivo human skin equivalent model shows that N-acetylcysteine promotes the keratinocyte differentiation and accelerates the re-epithelialization process. Our hydrogel dressing can be made into different formats for the healing of both flat and deep infected chronic wounds without contamination of the wound or needing other modalities such as photothermal irradiation.

Encapsulation of telmisartan inside insulinoma-cell-derived extracellular vesicles outperformed biomimetic nanovesicles in modulating the pancreatic inflammatory microenvironment.

Diabetes mellitus (DM) is a chronic metabolic condition, characterized by hyperglycaemia, oxidative imbalance, pancreatic β-cell death, and insulin insufficiency. Angiotensin II (Ang II) increases oxidative stress, inflammation, and apoptosis, and Ang II type 1 receptor (AT1R) blockers (ARBs) can ameliorate inflammatory response and oxidative stress. However, like other small-molecule drugs, free ARBs show poor in vivo efficacy and dose-limiting toxicities. Hence, in this study, we developed nano-formulations of telmisartan (TEL), an ARB, by encapsulating it inside a murine insulinoma cell-derived extracellular vesicle (nanoTEL) and a bio-mimetic lipid nanovesicle (lipoTEL). Both nano-formulations showed spherical morphology and sustained release of TEL. In vitro, nanoTEL restored oxidative equilibrium, attenuated reactive oxygen species levels, enhanced the uptake of glucose analogue, and increased the expression of glucose transporter protein 4 better than lipoTEL. In a streptozotocin-induced murine model of diabetes, nanoTEL lowered blood glucose levels, improved glucose tolerance, and promoted insulin synthesis and secretion significantly better than lipoTEL. Moreover, nanoTEL was found superior in ameliorating the pancreatic inflammatory microenvironment by regulating NF-κBp65, HIF-1α, and PPAR-γ expression; modulating IL-1β, IL-6, tumor necrosis factor-α, IL-10, and IL-4 levels and inducing the polarization of macrophage from M1 to M2. Further, nanoTEL administration induced angiogenesis and promoted the proliferation of pancreatic cells to restore the structural integrity of the islets of Langerhans more efficiently than lipoTEL. These findings collectively suggest that nanoTEL outperforms lipoTEL in restoring the function of pancreatic β-cells by modulating the pancreatic inflammatory microenvironment and show potential for the treatment of DM.

A PEDF peptide mimetic effectively relieves dry eye in a diabetic murine model by restoring corneal nerve, barrier, and lacrimal gland function

PurposeThe study investigated effectiveness of a novel PEDF peptide mimetic to alleviate dry eye-like pathologies in a Type I diabetic mouse model established using streptozotocin. MethodsMice were treated topically for 3–6 weeks with Ppx (a 17-mer PEDF mimetic) 2x/day or vehicle. Corneal sensitivity, tear film, epithelial and endothelial injury were measured using Cochet-Bonnet esthesiometer, phenol red cotton thread wetting, fluorescein sodium staining, and ZO1 expression, respectively. Inflammatory and parasympathetic nerve markers and activation of the MAPK/JNK pathways in the lacrimal glands were measured. ResultsDiabetic mice exhibited features of dry eye including reduced corneal sensation and tear secretion and increased corneal epithelium injury, nerve degeneration, and edema. Ppx reversed these pathologies and restored ZO1 expression and morphological integrity of the endothelium. Upregulation of IL-1β and TNFα, increased activation of P-38, JNK, and ERK, and higher levels of M3ACHR in diabetic lacrimal glands were also reversed by the peptide treatment. ConclusionThe study demonstrates that topical application of a synthetic PEDF mimetic effectively alleviates diabetes-induced dry eye by restoring corneal sensitivity, tear secretion, and endothelial barrier and lacrimal gland function. These findings have significant implications for the potential treatment of dry eye using a cost-effective and reproducible approach with minimal invasiveness and no obvious side effects.

Advantages and Limitations of Diabetic Bone Healing in Mouse Models: A Narrative Review.

Diabetes represents a major risk factor for impaired fracture healing. Type 2 diabetes mellitus is a growing epidemic worldwide, hence an increase in diabetes-related complications in fracture healing can be expected. However, the underlying mechanisms are not yet completely understood. Different mouse models are used in preclinical trauma research for fracture healing under diabetic conditions. The present review elucidates and evaluates the characteristics of state-of-the-art murine diabetic fracture healing models. Three major categories of murine models were identified: Streptozotocin-induced diabetes models, diet-induced diabetes models, and transgenic diabetes models. They all have specific advantages and limitations and affect bone physiology and fracture healing differently. The studies differed widely in their diabetic and fracture healing models and the chosen models were evaluated and discussed, raising concerns in the comparability of the current literature. Researchers should be aware of the presented advantages and limitations when choosing a murine diabetes model. Given the rapid increase in type II diabetics worldwide, our review found that there are a lack of models that sufficiently mimic the development of type II diabetes in adult patients over the years. We suggest that a model with a high-fat diet that accounts for 60% of the daily calorie intake over a period of at least 12 weeks provides the most accurate representation.