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The article presents a study on the relationship between bacterial load and the healing rate in neuropathic diabetic foot ulcers. Saline was used in flushing wound exudates and necrotic tissues were removed by local debridement. Staphylococcus (S.) aureus and S. epidermidis were the bacterial species present in the wound fluid. Base on the results, it was concluded that high bacterial load might contribute to impaired wound healing.

Endothelial dysfunction in macrovascular disease is well characterised by measurement of in vivo arterial blood flow and various biomarkers. In contrast, abnormalities of microvascular blood flow and biomarkers in the development of microvascular complications is less well documented. The aim of this study was to characterise microvascular blood flow and biomarkers in patients with type 2 diabetes with (D+ve Comps) and without (D-ve Comps) microvascular complications. We recruited 20 D+ve Comps [Age: 55 yrs (IQR 49 - 62), BMI: 32.6 ± 6.6 kg/m², Duration: 14.1 ± 6.2 yrs, HbA1c: 8.2% (IQR 7.5 - 9.2)], 20 D-ve Comps [Age: 58 yrs (IQR 54 - 64), BMI. 28.6 ± 4.8 kg/m², Duration: 5.8 ± 4.8 yrs, HbA1c: 6.4% (IQR 6.3 - 7.2)] and 20 age matched control subjects [Age: 56 yrs (IQR 47 - 61), BMI: 27.4 ± 4.0 kg/m²]. Patients with a history of macrovascular event were excluded. Blood was collected for biomarkers: C reactive protein (hsCRP), PAI-1 and the adhesion molecules sICAM and sVCAM. Skin blood flow (SkBF) of the forearm was measured using laser Doppler Velocimetry (LDV) combined with iontophoresis of acetycholine (ACh; endothelium dependent) and sodium nitroprusside (SNP; endothelium independent). Results are expressed as mean ± SD or median (interquartile range), and SkBF as fold increase from baseline. Both ACh and SNP responses progressively fall with development of diabetes and complications. There was a significant overall correlation between ACh and SNP responses (r=0.7, p<0.0001), but this relationship was more evident in control subjects and those with complications. We conclude that abnormalities of microvascular blood flow become progressively evident in parallel with the development of microvascular diabetic complications. This relationship was stronger than that pertaining to conventional biomarkers of vascular disease and inflammation. As both ACh and SNP responses became attenuated, the disturbance is not characteristic of endothelial dysfunction alone but could also be due to excessive quenching of NO or defective vascular smooth muscle response to NO, or to factors such as stiffness of the microvessels. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editors discuss various reports within the issue on topics including diabetes complications, post-surgical diabetic wounds, and amputation.

Extracellular matrix turnover is a prerequisite for normal wound healing, however this needs to be regulated as uncontrolled proteolysis is a pathogenic factor in the non-healing of wounds. The matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and transforming growth factor-β (TGF-β) are important regulators of this wound remodelling process but there is very limited data on their impact on wound healing rate (WHR) in diabetes. The aim of this study was to investigate the relationship of MMP -2 & -9, TIMP-1 and TGF-β with WHR in diabetic patients with foot ulcers. Diabetic patients with foot ulcers (Texas Grading 0 - 2 and stage A or B ; initial ulcer area: 315±345mm²) attending our Foot Clinic were studied at presentation and subsequent follow up. Forty two patients (M/F 31/11, age 60.5±10.1yrs, diabetes duration 14.5±7.5yrs) were enrolled. The ulcers were debrided and exudates removed with saline. Wound fluid was collected by absorption onto a sterile 1 cm² filter paper and recovered by immersion in PBS. The MMP-2&-9 (active and pro forms) were analysed by quantitative zymography; TIMP-1 and TGF-β by ELISA. WHR was determined by NIH Image analysis of wound tracings and calculated as % change in ulcer area over 28 days. There was no relationship between wound area at presentation and levels of MMP-2,-9, TIMP-1 or TGF-β. The MMPs decreased whilst TIMP-1 and TGF-β increased progressively with wound healing. The MMP-9 level and MMP-9/TIMP-1 ratio, particulary the pro-form, correlated inversely with WHR at 28 days (r= -0.48 and r= - 0.57 respectively, p<0.001) but this relationship was not evident for MMP-2, TIMP-1 or TGF-β. The MMP-9 level and MMP-9/TIMP-1 ratio were also lower when results were analysed with respect to the 16 patients who achieved complete wound healing at wk 12 versus the 26 that did not (proMMP-9 : 5.2+4.2 vs 8.7+5.0 ng/ml; proMMP-9/TIMP-1 : 7.9+6.8 vs 34.0+49.6, p<0.05). These findings suggest that a milieu with low MMP-9 is conducive to wound healing. This could be related to the fact that MMP-9 is derived mainly from inflammatory cells and a high level may indicate the persistence of inflammation. Whether this observation is causally related to our previous finding of high wound bacterial count impeding healing remains to be determined. Measurement of MMP-9 and TIMP-1 in wound fluid may help to identify ulcers at risk of poor healing. Supported by Diabetes Australia Research Trust [ABSTRACT FROM AUTHOR]

Aims: Delayed healing of diabetes-related foot ulcers (DRFUs) is associated with increased macrophage and matrix metalloproteinases (MMPs) at the wound site. Whether circulating monocyte phenotype and/or MMPs are altered in association with wound healing outcome is unknown, and was investigated in this study.Methods: Blood was obtained from 21 participants with DRFU, at initial visit (V1), week-4 (V2), and week-8 (V3) for measurement of monocyte number (CD14+), phenotype (CD16, CD163) and chemokine receptors (CCRs) by flow cytometry, and circulating MMPs and TIMP-1 by ELISA.Results: Six wounds healed during the study. At V1, non-classical CD16++ monocytes and MMP-3 were higher in healed vs unhealed (both p < 0.05). At V3, the increased %CD16++ persisted and %CCR2+ was decreased in healed, but no other monocyte markers nor MMP/TIMP differed between groups. Increased wound closure rate (WCR) at V3 correlated with increased %CD16++ monocytes and decreased MMP-2 at V1 or V1 + V2. Receiver operating characteristic (ROC) curves yielded an area-under-the-curve of %CD16++ at V1 of 0.78 to predict ulcer healing at V3.Conclusions: These results indicate that circulating monocyte phenotype and MMPs alter as DRFUs heal. The relationship of %CD16++ monocytes with WCR and ROC curve suggest a predictive role of %CD16++ monocytes for ulcer healing. [ABSTRACT FROM AUTHOR]

CD163, a scavenger receptor with anti-inflammatory function expressed exclusively on monocytes/macrophages, is dysregulated in cases of diabetes complications. This study aimed to characterize circulating CD163+ monocytes in the presence (D +Comps ) or absence (D -Comps ) of diabetes-related complications. RNA-sequencing and mass cytometry were conducted on CD163+ monocytes in adults with long-duration diabetes and D +Comps or D -Comps . Out of 10,868 differentially expressed genes identified between D +Comps and D -Comps , 885 were up-regulated and 190 were down-regulated with a ≥ 1.5-fold change. In D +Comps , 'regulation of centrosome cycle' genes were enriched 6.7-fold compared to the reference genome. MIR27A, MIR3648-1, and MIR23A , the most up-regulated and CD200R1 , the most down-regulated gene, were detected in D +Comps from the list of 75 'genes of interest'. CD163+ monocytes in D +Comps had a low proportion of recruitment markers CCR5, CD11b, CD11c, CD31, and immune regulation markers CD39 and CD86. A gene-protein network identified down-regulated TLR4 and CD11b as 'hub-nodes'. In conclusion, this study reports novel insights into CD163+ monocyte dysregulation in diabetes-related complications. Enriched centrosome cycle genes and up-regulated miRNAs linked to apoptosis, coupled with down-regulated monocyte activation, recruitment, and immune regulation, suggest functionally distinct CD163+ monocytes in cases of diabetes complications. Further investigation is needed to confirm their role in diabetes-related tissue damage.

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process., (© 2022. Crown.)

Recent scientific efforts have focused on the detrimental effects that obesity has on the metabolic function of skeletal muscles and whether exercise can improve this dysfunction. In this regard, adiponectin, with important metabolic functions (e.g. insulin-sensitizer and anti-inflammatory), has been recently described as a myokine that acts in an autocrine/paracrine manner. Earlier studies reported that muscle adiponectin could be induced by pro-inflammatory mediators (e.g. lipopolysaccharide), cytokines, and high-fat diets, providing a protective mechanism of this tissue against metabolic insults. However, when metabolic insults such as high-fat diets are sustained this protective response becomes dysregulated, making the skeletal muscle susceptible to metabolic impairments. Recent studies have suggested that exercise could prevent or even reverse this process. Considering that most scientific knowledge on adiponectin dysregulation in obesity is from the study of adipose tissue, the present review summarizes and discusses the literature available to date regarding the effects of obesity on skeletal muscle adiponectin induction, along with the potential effects of different exercise prescriptions on this response in an obesity context., (Copyright © 2019 Elsevier Inc. All rights reserved.)

People with diabetes mellitus have shorter telomeres compared with non-diabetic subjects. The aim of this study was to investigate an in-vitro model of telomere shortening under diabetes metabolic conditions. The mechanisms of the accelerated telomere length attrition and the potential telomere protective action of fenofibrate with related cellular mechanisms were also examined. Human dermal fibroblasts were passaged and cultured in normal (5.5 mM) or high (25 mM) D-glucose, across 7 days with hydrogen peroxide (H 2 O 2 ), glucosamine (GA), or glycated albumin (AGEs-BSA). Relative telomere length (RTL) was determined by qPCR. The expression of shelterin complex members which regulate telomere stability were measured by qRT-PCR and Western immunoblot. Culture in high glucose decreased RTL compared with normal glucose: H 2 O 2 and GA lowered the RTL after 7 days (each P < 0.05 vs untreated control), whereas AGEs-BSA had no effect compared with control-BSA. At day 7 the mRNA levels of most shelterin complex members, were induced by H 2 O 2 and to a lesser extent by GA. Trf1 and Trf2 protein were induced by H 2 O 2. Co-treatment with fenofibrate (100 μM) significantly attenuated the reduction in RTL caused by H 2 O 2 and GA and prevented Trf induction by H 2 O 2 . However knockdown of Trf1 and Trf2 expression using specific siRNA did not prevent H 2 O 2 effects to lower RTL, thus implicating factors other than these Trfs alone in the fenofibrate protection against the H 2 O 2 induction of RTL lowering. These in vitro findings demonstrate that diabetic conditions can induce telomere shortening and that fenofibrate has protective effects on telomere attrition, through as yet undefined mechanisms.

Many studies have shown effects of members of the CCN family on matrix synthesis and accumulation but few have examined degradative pathways. This scarcity of information is in part due to the lack of suitable model systems. Here we describe methods for making rhCCN2 and also for the preparation of a biosynthetically labeled matrix substrate that can be used to measure the effect of CCN on cellular or secreted degradative pathways.

Objective: To determine if white blood cell (WBC) telomeres are shorter in Type 1 diabetes (T1D) than in subjects without diabetes (non-DB), and shorter in T1D subjects with vs. without vascular complications; and to determine associations with vascular biomarkers., Research Design and Methods: WBC relative telomere length (RTL) was determined by quantitative PCR in a cross-sectional study of 140 non-DB and 199 T1D adults, including 128 subjects without vascular complications (T1DNoCx) and 71 subjects with vascular complications (T1DCx). Relationships of RTL with age, T1D duration, arterial elasticity, pulse pressure and vascular risk factors were determined., Results: RTL did not differ by gender within T1D and non-DB groups. Age-adjusted RTL was shorter in T1D vs. non-DB subjects (1.48±0.03 AU vs. 1.64±0.04 AU, p=0.002), but did not differ by T1D complication status (T1DNoCX 1.50±0.04 vs. T1DCX 1.46±0.05, p=0.50), nor correlate with arterial elasticity. Univariate analysis in T1D showed RTL correlated (inversely) with age r=-0.27, p=0.0001, T1D duration r=-0.16, p=0.03, and pulse pressure (r=-0.15, p=0.04), but not with HbA1c, BP, renal function (serum creatinine, ACR, eGFR), lipids, insulin sensitivity, inflammation (CRP, CAMs) or oxidative stress (OxLDL, OxLDL/LDL-C, MPO, PON-1). Multiple regression analysis showed independent determinants of RTL were age and T1D presence (r=0.29, p<0.0001)., Conclusions: In this cross-sectional study telomeres were shorter in T1D. RTL correlated inversely with T1D duration, but did not differ by complication status and weakly correlated with pulse pressure and vascular risk factors. Only age and T1D were independent determinants of RTL. Longitudinal studies are merited., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Connective tissue growth factor (CTGF), also known as CCN-2, is a cysteine-rich secreted protein that is involved in a range of biological processes, including regulation of cell growth and differentiation. Our previous in vitro studies have shown that CCN-2 inhibits adipocyte differentiation, although whether CCN-2 is regulated in vivo in adipogenesis is undetermined and was investigated in this study. C57BL/6 male mice were fed either standard laboratory chow (ND) or a diet high in fat (HFD; 45% fat) for 15 or 24 wk. HFD animals that gained >5 g in weight (termed HFD-fat) were insulin resistant and were compared with HFD-fed animals, which failed to gain weight (termed HFD-lean). HFD-fat mice had significantly increased CCN-2 mRNA levels in both the subcutaneous and epididymal fat pads, whereas CCN-2 mRNA was not induced in the epididymal site in HFD-lean mice. Also in HFD-fed animals, epididymal CCN-2 mRNA correlated positively with key genes involved in adipocyte differentiation, adiponectin and PPARγ (P < 0.001 and P < 0.002, respectively). Additionally, epididymal CCN-2 mRNA correlated positively with two markers of tissue turnover, PAI-1 in HFD-fat mice only and TIMP-1, but only in the HFD-lean mice. Collectively, these findings suggest that CCN-2 plays a role in adipocyte differentiation in vivo and thus in the pathogenesis of obesity linked with insulin resistance.