Upregulation of Anti-Angiogenic miR-106b-3p Correlates Negatively with IGF-1 and Vascular Health Parameters in a Model of Subclinical Cardiovascular Disease: Study with Metformin Therapy.

Well-controlled type 1 diabetes mellitus (T1DM) is regarded as a model of subclinical cardiovascular disease (CVD), characterized by inflammation and adverse vascular health. However, the underlying mechanisms are not fully understood. We investigated insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) levels, their correlation to miR-106b-3p expression in a subclinical CVD model, and the cardioprotective effect of metformin. A total of 20 controls and 29 well-controlled T1DM subjects were studied. Plasma IGF-1, IGFBP-3 levels, and miR-106b-3p expression in colony-forming unit-Hills were analyzed and compared with vascular markers. miR-106b-3p was upregulated in T1DM (p < 0.05) and negatively correlated with pro-angiogenic markers CD34+/100-lymphocytes (p < 0.05) and IGF-1 (p < 0.05). IGF-1 was downregulated in T1DM (p < 0.01), which was associated with increased inflammatory markers TNF-α, CRP, and IL-10 and reduced CD34+/100-lymphocytes. IGFBP-3 had no significant results. Metformin had no effect on IGF-1 but significantly reduced miR-106b-3p (p < 0.0001). An Ingenuity Pathway analysis predicted miR-106b-3p to inhibit PDGFA, PIK3CG, GDNF, and ADAMTS13, which activated CVD. Metformin was predicted to be cardioprotective by inhibiting miR-106b-3p. In conclusion: Subclinical CVD is characterized by a cardio-adverse profile of low IGF-1 and upregulated miR-106b-3p. We demonstrated that the cardioprotective effect of metformin may be via downregulation of upregulated miR-106b-3p and its effect on downstream targets.

Decoding of miR-7-5p in Colony Forming Unit-Hill Colonies as a Biomarker of Subclinical Cardiovascular Disease-A MERIT Study.

Colony forming unit-Hill (CFU-Hill) colonies were established to serve as a sensitive biomarker for vascular health. In animals, the overexpression of miR-7-5p was shown to be pro-atherogenic and associated with increased cardiovascular disease (CVD) risk. In a MERIT study, we aimed to explore the role of miR-7-5p expression in CFU-Hill colonies in type 1 diabetes mellitus (T1DM) and the effect of metformin in subclinical CVD. The expression of miR-7-5p in CFU-Hill colonies in 29 T1DM subjects without CVD and 20 healthy controls (HC) was measured. Metformin was administered to T1DM subjects for eight weeks. MiR-7-5p was upregulated in T1DM whereas metformin reduced it to HC levels. MiR-7-5p was positively correlated with c-reactive protein, and C-X-C motif chemokine ligand 10. The receiver operating characteristic curve revealed miR-7-5p as a biomarker of CVD, and upregulated miR-7-5p, defining subclinical CVD at a HbA1c level of 44.3 mmol/mol. Ingenuity pathway analysis predicted miR-7-5p to inhibit the mRNA expression of Krüppel-like factor 4, epidermal growth factor receptor, insulin-like growth factor 1 receptor, v-raf-1 murine leukemia viral oncogene homolog 1 and insulin receptor substrate ½, and insulin receptor, while metformin activated these miRNAs via transforming growth factor-ß1 and Smad2/3. We proved the pro-atherogenic effect of miR-7-5p that maybe used as a prognostic biomarker.

Deciphering the Role of miR-200c-3p in Type 1 Diabetes (Subclinical Cardiovascular Disease) and Its Correlation with Inflammation and Vascular Health.

Uncomplicated type 1 diabetes (T1DM) displays all features of subclinical cardiovascular disease (CVD) as is associated with inflammation, endothelial dysfunction and low endothelial progenitor cells. MiR-200c-3p has been shown in animal tissues to be pro-atherogenic. We aimed to explore the role of miR-200c-3p in T1DM, a model of subclinical CVD. 19 samples from T1DM patients and 20 from matched controls (HC) were analyzed. MiR-200c in plasma and peripheral blood mononuclear cells (PBMCs) was measured by real-time quantitative polymerase chain reaction. The results were compared with the following indices of vascular health: circulating endothelial progenitor cells, (CD45dimCD34+VEGFR-2+ or CD45dimCD34+CD133+) and proangiogenic cells (PACs). MiR-200c-3p was significantly downregulated in PBMCs but not in plasma in T1DM. There was a significant negative correlation between the expression of miR-200c-3p and HbA1c, interleukin-7 (IL-7), vascular endothelial growth factor-C (VEGF-C), and soluble vascular cell adhesion molecule-1, and a positive correlation with CD45dimCD34+VEGFR-2+, CD45dimCD34+CD133+ and PACs. Receiver operating curve analyses showed miR-200c-3p as a biomarker for T1DM with significant downregulation of miR-200c-3p, possibly defining subclinical CVD at HbA1c > 44.8 mmol/mol (6.2%). In conclusion, downregulated miR-200c-3p in T1DM correlated with diabetic control, VEGF signaling, inflammation, vascular health and targeting VEGF signaling, and may define subclinical CVD. Further prospective studies are necessary to validate our findings in a larger group of patients.

Upregulated miR-18a-5p in Colony Forming Unit-Hill's in Subclinical Cardiovascular Disease and Metformin Therapy; MERIT Study.

Colony forming unit-Hill's (CFU-Hill's) colonies are hematopoietic-derived cells that participate in neovasculogenesis and serve as a biomarker for vascular health. In animals, overexpression of miR-18a-5p was shown to be pro-atherogenic. We had shown that well-controlled type 1 diabetes mellitus (T1DM) is characterized by an inflammatory state, endothelial dysfunction, and reduced number of CFU-Hill's, a model of subclinical cardiovascular disease (CVD). MERIT study explored the role of miR-18a-5p expression in CFU-Hill's colonies in T1DM, and the cardioprotective effect of metformin in subclinical CVD. In T1DM, miR-18a-5p was significantly upregulated whereas metformin reduced it to HC levels. MiR-18a-5p was inversely correlated with CFU-Hill's colonies, CD34+, CD34+CD133+ cells, and positively with IL-10, C-reactive protein, vascular endothelial growth factor-D (VEGF-D), and thrombomodulin. The receiver operating characteristic curve demonstrated, miR-18a-5p as a biomarker of T1DM, and upregulated miR-18a-5p defining subclinical CVD at HbA1c of 44.5 mmol/mol (pre-diabetes). Ingenuity pathway analysis documented miR-18a-5p inhibiting mRNA expression of insulin-like growth factor-1, estrogen receptor-1, hypoxia-inducible factor-1α cellular communication network factor-2, and protein inhibitor of activated STAT 3, whilst metformin upregulated these mRNAs via transforming growth factor beta-1 and VEGF. We confirmed the pro-atherogenic effect of miR-18a-5p in subclinical CVD and identified several target genes for future CVD therapies.

Subclinical Thyrotoxicosis and Cardiovascular Risk: Assessment of Circulating Endothelial Progenitor Cells, Proangiogenic Cells, and Endothelial Function.

Background: Subclinical thyrotoxicosis (SCT) is defined by low or undetectable thyroid-stimulating hormones and normal thyroid hormones. The treatment of SCT is uncertain despite being associated with increased cardiovascular risk (CVR) and mortality. Circulating endothelial progenitor cells (cEPCs) and circulating angiogenic cells (CACs) have been found to be reduced in conditions with CVR. We aimed to evaluate whether endothelial function and cEPC and CAC counts were reduced in SCT and to study the in vitro effect of triiodothyronine (T3) on proangiogenic cell (PAC) function from young healthy controls. Methods: cEPCs (quantified by flow cytometry, 20 SCT/20 controls), CACs following in vitro cultures (15 SCT/14 controls), paracrine function of CACs, endothelial function by flow-mediated dilation (FMD, 9 SCT/9 controls), and the effect of T3 on apoptosis and endothelial nitric oxide synthase (eNOS) expression in PACs were studied. Results: p < 0.001, CD133+/VEGFR-2+ 0.4 (0.0-0.7) vs. 0.6 (0.0-4.6), p = 0.009, CD34+/VEGFR-2+ 0.3 (0.0-1.0) vs. 0.7 (0.1-4.9), p = 0.002; while CAC count was similar. SCT predicted a lower cEPC count after adjustment for conventional CVR factors. FMD was lower in SCT subjects versus controls (% mean ± SD, 2.7 ± 2.3 vs. 6.1 ± 2.3, p = 0.005). In vitro studies showed T3 increased early apoptosis and reduced eNOS expression in PACs. Conclusions: In conclusion, SCT is associated with reduced cEPC count and FMD, confirming increased CVR in SCT. Future outcome trials are required to examine if treatment of this subclinical hyperactive state improves cardiovascular outcome. Clinical Trial Registration: http://www.controlled-trials.com/isrctn/, identifier ISRCTN70334066.

Radioactive Iodine for the Treatment of Subclinical Thyrotoxicosis Grade 1 and 2: Outcome of up to 18-Year Follow Up.

Background: Subclinical thyrotoxicosis (SCT) is associated with significant morbidity and mortality, specifically increased risk of atrial fibrillation and cardiovascular death. The management is ill-defined due to the scarcity of randomised controlled studies. Some clinicians recommend radioiodine (RAI) treatment however its long-term outcome is unknown. Therefore, further data is needed to provide robust evidence-based guidelines. Methods: A prospective, single-protocol analysis of the outcome of SCT patients (Grade 1; 0.1-0.4 mIU/L and Grade 2; <0.1 mIU/L) treated with mean dose of 427 MBq of I131, followed up for up to 18 years. Thyroid function tests were measured at 4-6 weeks, 3-, 6-, and 12-months post-RAI, and annually thereafter. Cure was defined as achieving a euthyroid/hypothyroid state. Results: Seventy-eight patients with a median age of 68 years (range 36-84) and varying aetiology [55 toxic multinodular goitre (TMNG), 10 toxic nodule (TN) and 13 Graves' disease (GD)] were followed up for a median period of 7.5 years (range 1-18). The cure rate was 100%. The rates of hypothyroidism in TMNG, TN and GD were 23.6%, 30% and 38.5% respectively. The median time to hypothyroidism was 6 and 12 months in GD and TMNG/TN respectively. No differences in outcome between Grade 1 versus Grade 2 were observed. Conclusion: RAI using single mean dose of 427 MBq is effective and safe, irrespective of aetiology or grade of TSH suppression. GD patients become hypothyroid within the first year, whilst TMNG/TN for up to 9-years. Thus after 12 months of follow up, annual thyroid function monitoring is advised.

The prevalence and significance of nonuniform thyroid radio-isotope uptake in patients with Graves' disease.

OBJECTIVE: To evaluate the prevalence and clinical significance of nonuniform technetium (99m Tc) uptake among patients with Graves' disease (GD). DESIGN, PATIENTS AND MEASUREMENTS: Patients with GD, referred between July 2005 and March 2018, had Tc99 - uptake scans and TSH-receptor antibody (TRAb) measured before antithyroid drug (ATD) therapy. Risk of relapse after ATD cessation was monitored until June 2021 and compared between GD patients based on uptake patterns. RESULTS: Of the 276 GD patients (mean age, 49.8 years; 84% female), 25 (9.0%) had nonuniform Tc99 uptake. At diagnosis, individuals with nonuniform uptake were older (mean age of 61.8 vs. 48.5 years, p < .001), had lower mean thyroid hormone levels (free thyroxine: 36.3 vs. 45.4 pmol/L, p = .04 and free triiodothyronine: 10.0 vs. 17.8 pmol/L, p < .001) and median TRAb levels (4.2 vs. 6.6 U/L, p = .04) compared with those with a uniform uptake. Older age was a significant predictor for the presence of nonuniform uptake in GD patients; odds ratio (95% confidence intervals) of 1.07 (1.03 - 1.10). The risk of relapse was similar in both groups after a median (IQR) follow-up of 41 (13-74) months after ATD cessation (56.0% vs. 46.3%, respectively); hazard ratio (95% confidence intervals) of 1.74 (0.96-3.15). CONCLUSIONS: Nonuniform radio-isotope uptake is seen in 1 in 11 patients with GD which could be misdiagnosed as toxic multinodular goitre if TRAb levels are not measured. Treatment of GD patients with nonuniform radio-isotope uptake with ATD therapy as first-line appears to be equally effective as compared with those with uniform uptake. TRAb testing should be the main diagnostic test for patients with suspected GD with radio-labelled uptake scans being reserved for those who are TRAb negative.

Upregulated anti-angiogenic miR-424-5p in type 1 diabetes (model of subclinical cardiovascular disease) correlates with endothelial progenitor cells, CXCR1/2 and other parameters of vascular health.

BACKGROUND: In spite of clinical progress, cardiovascular disease (CVD) remains the predominant cause of mortality worldwide. Overexpression studies in animals have proven miR-424-5p to have anti-angiogenic properties. As type 1 diabetes mellitus (T1DM) without CVD displays endothelial dysfunction and reduced circulating endothelial progenitor cells (cEPCs), it offers a model of subclinical CVD. Therefore, we explored miR-424-5p, cytokines and vascular health in T1DM. METHODS: Twenty-nine well-controlled T1DM patients with no CVD and 20-matched controls were studied. Cytokines IL8, TNF-α, IL7, VEGF-C, cEPCs/CD45dimCD34+CD133+ cells and ex-vivo proangiogenic cells (PACs)/fibronectin adhesion assay (FAA) were measured. MiR-424-5p in plasma and peripheral blood mononuclear cells (PBMC) along with mRNAs in PBMC was evaluated. RESULTS: We found an elevation of IL7 (p = 0.008), IL8 (p = 0.003), TNF-α (p = 0.041), VEGF-C (p = 0.013), upregulation of mRNA CXCR1 (p = 0.009), CXCR2 (p < 0.001) and reduction of cEPCs (p < 0.001), PACs (p < 0.001) and FAA (p = 0.017) in T1DM. MiR-424-5p was upregulated in T1DM in PBMC (p < 0.001). MiR-424-5p was negatively correlated with cEPCs (p = 0.006), PACs (p = 0.005) and FAA (p < 0.001) and positively with HbA1c (p < 0.001), IL7 (p = 0.008), IL8 (p = 0.017), VEGF-C (p = 0.007), CXCR1 (p = 0.02) and CXCR2 (p = 0.001). ROC curve analyses showed (1) miR-424-5p to be a biomarker for T1DM (p < 0.001) and (2) significant upregulation of miR-424-5p, defining subclinical CVD, occurred at HbA1c of 46.5 mmol/mol (p = 0.002). CONCLUSION: We validated animal research on anti-angiogenic properties of miR-424-5p in T1DM. MiR-424-5p may be a biomarker for onset of subclinical CVD at HbA1c of 46.5 mmol/mol (pre-diabetes). Thus, miR-424-5p has potential use for CVD monitoring whilst anti-miR-424-5p-based therapies may be used to reduce CVD morbidity/mortality in T1DM.

MiR-126, IL-7, CXCR1/2 receptors, inflammation and circulating endothelial progenitor cells: The study on targets for treatment pathways in a model of subclinical cardiovascular disease (type 1 diabetes mellitus).

BACKGROUND: Type 1 diabetes (T1DM) is associated with premature cardiovascular disease (CVD) and a pro-inflammatory state whilst the proangiogenic miR-126-3p/-5p may play a role in CVD. Animal studies established miR-126 to be pro-angiogenic. We hypothesised miR-126-3p/-5p are reduced in T1DM whilst pro-inflammatory cytokines are increased. METHODS: 29 well controlled, T1DM patients without CVD and 20 healthy controls (HCs) were studied. MiR-126-3p/-5p were assayed in plasma and peripheral blood mononuclear cells (PBMCs) whilst Chemokine C-X-C Receptor 1/2 (CXCR1/2) mRNA in PBMCs by real-time quantitative PCR. Cytokines were assayed by the Mesoscale Discovery. Ingenuity Pathway Analysis (IPA) was used to predict target genes, cellular functions and pathological states regulated by miR-126-3p/-5p. IPA generated both direct and indirect causations between different targets and analysed whether these effects would be inhibitory or stimulatory based on the published evidence. RESULTS: T1DM patients had a relatively good diabetic control (HbA1c = 7.4 ± 0.7% or 57.3 ± 7.6 mmol/mol). Homeostatic cytokine IL-7, pro-inflammatory cytokines IL-8 and TNF-α, and vascular endothelial growth factor-C (VEGF-C) were increased in T1DM, versus HCs; p = 0.008, p = 0.003, p = 0.041 and p = 0.013 respectively. MiR-126-5p was significantly upregulated in PBMCs in T1DM versus HCs; p = 0.01, but not in plasma. MiR-126-3p was unchanged. CXCR1/2 were elevated in T1DM versus HCs; p = 0.009 and p < 0.001 respectively. MiR-126-5p was positively correlated with CXCR1/2, and with HbA1c whilst negatively correlated with circulating endothelial progenitor cells (CD34+CD133+CD45dim) and fibronectin adhesion assay in a combined group of T1DM patients and HCs; p = 0.028 p = 0.049 p = 0.035 p = 0.047 and p = 0.004 respectively. IPA predicted miR-126-5p to be anti-inflammatory through the inhibition of chemokine C-C motif ligand 27, chymotrypsin-like elastase 2A and IL-7, whilst miR-126-3p had no direct anti-inflammatory effect. Simultaneously IPA predicted IL-7 as the most upstream cytokine target. CONCLUSIONS: T1DM without apparent CVD or diabetic complications is an inflammatory state characterised not only by raised pro-inflammatory cytokines but also by increased receptor CXCR1/2 and miR-126-5p. MiR-126-5p upregulation may represent a compensatory response. Pro-miR-126-5p therapies or anti-IL-7 therapies may be a new option to reduce both inflammation and CVD risk in T1DM. Further research is required in a large prospective study in patients with T1DM.

The Role of miR-342 in Vascular Health. Study in Subclinical Cardiovascular Disease in Mononuclear Cells, Plasma, Inflammatory Cytokines and PANX2.

Cardiovascular disease (CVD) correlates with inflammation and a reduction in circulating endothelial progenitor cells (cEPCs). Recently, CVD was shown to be the main cause of mortality in individuals with type 1 diabetes (T1DM). In animals, miR-342 was shown to exert an anti-inflammatory effect in CVD. Hypothesis: miR-342-3p/-5p are downregulated in subclinical CVD (T1DM), whereas inflammatory cytokines are upregulated. We studied miR -342 -3p/5p in plasma/peripheral blood mononuclear cells (PBMCs) in 29 T1DM and 20 controls (HC). Vascular health was measured by fibronectin adhesion assay (FAA), cEPCs (CD45dimCD34+133+ cells) and by assessing inflammation and tissue inhibition of metalloproteases (TIMP-1). In T1DM IL-7, IL-8, TNFα and VEGF-C were increased in plasma. MiR-342-3p/-5p were downregulated in PBMCs in T1DM, but not in plasma. PANX2, chemokine receptors CXCR1/2 mRNAs, were increased in PBMCs in T1DM. MiR-342-3p was negatively correlated with TIMP-1, IL-6, IL-8, TNF-α, HbA1c and CXCR2, whilst miR-342-5p was negatively correlated with TIMP-1, IL-6, IL-8 and HbA1c. There was a positive correlation among miR-342-3p, FAA and cEPCs, and between miR-342-5p and cEPCs. ROC curve analyses showed significant downregulation of miR-342-3p/-5p at HbA1c > 46.45 mmol/mol, indicating their potential as biomarkers for subclinical CVD. Our findings validated animal studies and confirmed the proangiogenic properties of miR-342-3p/-5p. MiR-342-3p/-5p-based intervention or monitoring may prove to be beneficial in managing CVD.

Age May Influence the Impact of TRAbs on Thyroid Function and Relapse-Risk in Patients With Graves Disease.

CONTEXT: Thyrotropin receptor antibodies (TRAbs) play a crucial role in the pathogenesis of Graves disease (GD). However, factors that influence the association of TRAbs with thyroid hormones and relapse risk in GD remain unclear. OBJECTIVE: We investigated the associations of TRAbs at diagnosis with thyroid hormones and relapse risk and potential factors that can influence these associations in GD. DESIGN AND SETTING: A prospective study in an endocrine center in England. PATIENTS AND MAIN OUTCOME MEASURES: Three hundred eighty-four consecutive patients with GD who had measurements of TRAbs and thyroid hormones at diagnosis. The association of TRAbs with thyroid hormones and relapse risk was assessed through linear regression and Cox proportional hazard models, adjusted for confounders. RESULTS: TRAbs were nonlinearly associated with thyroid hormones, following a curve with an initial positive slope and a subsequent flattening (P < 0.0001). Higher TRAbs were associated with greater relapse risk [hazard ratio (HR), 1.05 (95% CI, 1.02 to 1.08) per 1-U/L increase]. These associations were modified by age, but not by sex, race, smoking, or thyroid peroxidase antibody levels. In younger participants, increasing TRAbs were associated with higher thyroid hormones and greater relapse risk [HR, 1.13 (95% CI, 1.04 to 1.23) per 1-U/L increase]. In older participants, TRAbs were not associated with thyroid hormones or relapse risk [HR, 0.99 (95% CI, 0.93 to 1.05) per 1-U/L increase. CONCLUSIONS: In GD, age can influence the effect of TRAbs on thyroid function and relapse risk. TRAbs at diagnosis have better predictive value in younger patients with GD.

Anti-Angiogenic miR-222, miR-195, and miR-21a Plasma Levels in T1DM Are Improved by Metformin Therapy, Thus Elucidating Its Cardioprotective Effect: The MERIT Study.

Type 1 diabetes (T1DM) is associated with increased cardiovascular disease (CVD) and reduced life expectancy. We thus hypothesized that anti-angiogenic miRs are increased in T1DM, and the cardioprotective effect of metformin is mediated via reducing those miRs. In an open label, case-controlled study, 23 T1DM patients without CVD were treated with metformin for eight weeks (TG), matched with nine T1DM patients on standard treatment (SG) and 23 controls (CG). Plasma miR-222, miR-195, miR-21a and miR-126 were assayed by real-time RT-qPCR. The results were correlated with: endothelial function (RHI), circulating endothelial progenitor cells (cEPCs) (vascular repair marker, CD45dimCD34⁺VEGFR2⁺ cells) and circulating endothelial cells (cECs) (vascular injury marker, CD45dimCD34⁺CD133⁻CD144⁺ cells). miR-222, miR-195 and miR-21a were higher in T1DM than CG; p = 0.009, p < 0.0001, p = 0.0001, respectively. There was an inverse correlation between logmiR-222 and logRHI (p < 0.05) and a direct correlation between logmiR-222 and logCD34⁺ (p < 0.05) in TG. Metformin reduced miR-222, miR-195 and miR-21a levels in TG; p = 0.007, p = 0.002 p = 0.0012, respectively. miRs remained unchanged in SG. miR-126 was similar in all groups. There was a positive association between changes in logmiR-222 and logcECs after metformin in TG (p < 0.05). Anti-angiogenic miRs are increased in T1DM. Metformin has cardioprotective effects through downregulating miR-222, miR-195 and miR-21a, beyond improving glycemic control.

Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia.

Cardiovascular disease is the leading cause of morbidity/mortality worldwide. Metformin is the first therapy offering cardioprotection in type 2 diabetes and non-diabetic animals with unknown mechanism. We have shown that metformin improves angiogenesis via affecting expression of growth factors/angiogenic inhibitors in CD34⁺ cells under hyperglycemia-hypoxia. Now we studied the direct effect of physiological dose of metformin on human umbilical vein endothelial cells (HUVEC) under conditions mimicking hypoxia-hyperglycemia. HUVEC migration and apoptosis were studied after induction with euglycemia or hyperglycemia and/or CoCl2 induced hypoxia in the presence or absence of metformin. HUVEC mRNA was assayed by whole transcript microarrays. Genes were confirmed by qRT-PCR, proteins by western blot, ELISA or flow cytometry. Metformin promoted HUVEC migration and inhibited apoptosis via upregulation of vascular endothelial growth factor (VEGF) receptors (VEGFR1/R2), fatty acid binding protein 4 (FABP4), ERK/mitogen-activated protein kinase signaling, chemokine ligand 8, lymphocyte antigen 96, Rho kinase 1 (ROCK1), matrix metalloproteinase 16 (MMP16) and tissue factor inhibitor-2 under hyperglycemia-chemical hypoxia. Therefore, metformin's dual effect in hyperglycemia-chemical hypoxia is mediated by direct effect on VEGFR1/R2 leading to activation of cell migration through MMP16 and ROCK1 upregulation, and inhibition of apoptosis by increase in phospho-ERK1/2 and FABP4, components of VEGF signaling cascades.

Metformin improves circulating endothelial cells and endothelial progenitor cells in type 1 diabetes: MERIT study.

BACKGROUND: Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. METHODS: This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45(dim)CD34(+)VEGFR-2(+) and CD45(dim)CD133(-)CD34(+)CD144(+) respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill's colonies). RESULTS: At baseline TG had lower cEPCs, PACs, CFU-Hills' colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill's colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill's colonies with cECs. CONCLUSIONS: Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill's colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132.

The Natural History of Subclinical Hyperthyroidism in Graves' Disease: The Rule of Thirds.

BACKGROUND: There is little information regarding the natural history of subclinical hyperthyroidism (SH) due to Graves' disease (GD). METHODS: A prospective analysis was conducted of patients with SH due to GD between 2007 and 2013 with at least 12 months of follow-up. SH was diagnosed if serum thyrotropin (TSH) was below the laboratory reference range (0.4-4.0 mIU/L) and when thyroid hormones were normal. GD was confirmed by either a raised TSH receptor antibody (TRAb) level or uniform uptake on Technetium scan. RESULTS: Forty-four patients (89% female, 16% current smokers, and 5% with active Graves' orbitopathy) were diagnosed with SH due to GD. Over the follow-up period (median 32 months), approximately one third (34%) of the cohort progressed to overt hyperthyroidism, one third (34%) normalized their thyroid function, slightly less than one third (30%) remained in the SH state, while one person became hypothyroid. Multivariate regression analysis showed that older age and positive antithyroid peroxidase (TPO) antibody status had a positive association with risk of progression to overt hyperthyroidism, with hazard ratios of 1.06 ([confidence interval (CI) 1.02-1.10], p < 0.01) per year and 10.15 ([CI 1.83-56.23], p < 0.01), respectively, independent of other risk factors including, smoking, TRAb levels at diagnosis, and sex. CONCLUSIONS: A third each of patients with SH due to GD progress, normalize, or remain in the SH state. Older people and those with positive anti-TPO antibodies have a higher risk of progression of the disease. These novel data need to be verified and confirmed in larger cohorts and over longer periods of follow-up.

Metformin improves the angiogenic potential of human CD34⁺ cells co-incident with downregulating CXCL10 and TIMP1 gene expression and increasing VEGFA under hyperglycemia and hypoxia within a therapeutic window for myocardial infarction.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with diabetes mellitus (DM). To identify the most effective treatment for CVD, it is paramount to understand the mechanism behind cardioprotective therapies. Although metformin has been shown to reduce CVD in Type-2 DM clinical trials, the underlying mechanism remains unexplored. CD34(+) cell-based therapies offer a new treatment approach to CVD. The aim of this study was to investigate the effect of metformin on the angiogenic properties of CD34(+) cells under conditions mimicking acute myocardial infarction in diabetes. METHODS: CD34(+) cells were cultured in 5.5 or 16.5 mmol/L glucose ± 0.01 mmol/L metformin and then additionally ± 4 % hypoxia. The paracrine function of CD34(+) cell-derived conditioned medium was assessed by measuring pro-inflammatory cytokines, vascular endothelial growth factor A (VEGFA), and using an in vitro tube formation assay for angiogenesis. Also, mRNA of CD34(+) cells was assayed by microarray and genes of interest were validated by qRT-PCR. RESULTS: Metformin increased in vitro angiogenesis under hyperglycemia-hypoxia and augmented the expression of VEGFA. It also reduced the angiogenic-inhibitors, chemokine (C-X-C motif) ligand 10 (CXCL10) and tissue inhibitor of metalloproteinase 1 (TIMP1) mRNAs, which were upregulated under hyperglycemia-hypoxia. In addition metformin, increased expression of STEAP family member 4 (STEAP4) under euglycemia, indicating an anti-inflammatory effect. CONCLUSIONS: Metformin has a dual effect by simultaneously increasing VEGFA and reducing CXCL10 and TIMP1 in CD34(+) cells in a model of the diabetic state combined with hypoxia. Therefore, these angiogenic inhibitors are promising therapeutic targets for CVD in diabetic patients. Moreover, our data are commensurate with a vascular protective effect of metformin and add to the understanding of underlying mechanisms.

Linkage Analysis in Autoimmune Addison's Disease: NFATC1 as a Potential Novel Susceptibility Locus.

BACKGROUND: Autoimmune Addison's disease (AAD) is a rare, highly heritable autoimmune endocrinopathy. It is possible that there may be some highly penetrant variants which confer disease susceptibility that have yet to be discovered. METHODS: DNA samples from 23 multiplex AAD pedigrees from the UK and Norway (50 cases, 67 controls) were genotyped on the Affymetrix SNP 6.0 array. Linkage analysis was performed using Merlin. EMMAX was used to carry out a genome-wide association analysis comparing the familial AAD cases to 2706 UK WTCCC controls. To explore some of the linkage findings further, a replication study was performed by genotyping 64 SNPs in two of the four linked regions (chromosomes 7 and 18), on the Sequenom iPlex platform in three European AAD case-control cohorts (1097 cases, 1117 controls). The data were analysed using a meta-analysis approach. RESULTS: In a parametric analysis, applying a rare dominant model, loci on chromosomes 7, 9 and 18 had LOD scores >2.8. In a non-parametric analysis, a locus corresponding to the HLA region on chromosome 6, known to be associated with AAD, had a LOD score >3.0. In the genome-wide association analysis, a SNP cluster on chromosome 2 and a pair of SNPs on chromosome 6 were associated with AAD (P <5x10-7). A meta-analysis of the replication study data demonstrated that three chromosome 18 SNPs were associated with AAD, including a non-synonymous variant in the NFATC1 gene. CONCLUSION: This linkage study has implicated a number of novel chromosomal regions in the pathogenesis of AAD in multiplex AAD families and adds further support to the role of HLA in AAD. The genome-wide association analysis has also identified a region of interest on chromosome 2. A replication study has demonstrated that the NFATC1 gene is worthy of future investigation, however each of the regions identified require further, systematic analysis.

The inflammation, vascular repair and injury responses to exercise in fit males with and without Type 1 diabetes: an observational study.

BACKGROUND: Type 1 diabetes is associated with raised inflammation, impaired endothelial progenitor cell mobilisation and increased markers of vascular injury. Both acute and chronic exercise is known to influence these markers in non-diabetic controls, but limited data exists in Type 1 diabetes. We assessed inflammation, vascular repair and injury at rest and after exercise in physically-fit males with and without Type 1 diabetes. METHODS: Ten well-controlled type 1 diabetes (27 ± 2 years; BMI 24 ± 0.7 kg.m(2); HbA1c 53.3 ± 2.4 mmol/mol) and nine non-diabetic control males (27 ± 1 years; BMI 23 ± 0.8 kg.m(2)) matched for age, BMI and fitness completed 45-min of running. Venous blood samples were collected 60-min before and 60-min after exercise, and again on the following morning. Blood samples were processed for TNF-α using ELISA, and circulating endothelial progenitor cells (cEPCs; CD45(dim)CD34(+)VEGFR2(+)) and endothelial cells (cECs; CD45(dim)CD133(-)CD34(+)CD144(+)) counts using flow-cytometry. RESULTS: TNF-α concentrations were 4-fold higher at all-time points in Type 1 diabetes, when compared with control (P < 0.001). Resting cEPCs were similar between groups; after exercise there was a significant increase in controls (P = 0.016), but not in Type 1 diabetes (P = 0.202). CEPCs peaked the morning after exercise, with a greater change in controls vs. Type 1 diabetes (+139 % vs. 27 %; P = 0.01). CECs did not change with exercise and were similar between groups at all points (P > 0.05). Within the Type 1 diabetes group, the delta change in cEPCS from rest to the following morning was related to HbA1c (r = -0.65, P = 0.021) and TNF-α (r = -0.766, P = 0.005). CONCLUSIONS: Resting cEPCs and cECs in Type 1 diabetes patients with excellent HbA1c and high physical-fitness are comparable to healthy controls, despite eliciting 4-fold greater TNF-α. Furthermore, Type 1 diabetes patients appear to have a blunted post-exercise cEPCs response (vascular repair), whilst a biomarker of vascular injury (cECs) remained comparable to healthy controls.

Levothyroxine improves abnormal cardiac bioenergetics in subclinical hypothyroidism: a cardiac magnetic resonance spectroscopic study.

CONTEXT: It is well established that subclinical hypothyroidism (SCH) is associated with mild cardiac dysfunction, but it is unknown whether there is an underlying impairment of cardiac bioenergetic function. OBJECTIVE: The objective of the study was to quantify the cardiac phosphocreatine to adenosine triphosphate ratio (PCr to ATP) in SCH, compared with healthy controls, and to measure the effect of 6 months of levothyroxine treatment. DESIGN AND SETTING: This was a 6-month, prospective, case-controlled interventional study. PARTICIPANTS AND MAIN OUTCOME MEASURES: The PCr to ATP ratio was measured using phosphorus-31 magnetic resonance spectroscopy in subjects with SCH at baseline and after levothyroxine therapy (1.6 µg/kg · d) and compared with age- and gender-matched euthyroid controls. All subjects were free of overt heart disease. RESULTS: Twenty-one subjects with SCH (normal free T4 and serum TSH between 4.1 and 10 mIU/L) and 17 controls were matched for age (mean age 40.5 vs 43.3 y) and sex (females 81% vs 82%) but differed in mean TSH (6.5 vs 2.1 mIU/L, P < .001). At baseline the mean (± SD) PCr to ATP ratio in SCH was lower than in controls (1.80 ± 0.26 vs 2.07 ± 0.20, P = .001). In the 16 subjects studied after levothyroxine treatment, the PCr to ATP ratio improved (from 1.74 ± 0.24 to 1.91 ± 0.26, P = .004) and approached controls (borderline loss of significance, P = .051). On multivariate analysis, SCH was independently associated with a reduced PCr to ATP ratio, even after adjusting for confounding variables (body mass index and fasting glucose) (P = .001). CONCLUSION: Our results demonstrate early cardiac bioenergetic impairment in SCH, which is reversible with levothyroxine therapy. This mechanistic insight provides justification for longitudinal trials to determine whether improvement in bioenergetic function improves cardiovascular outcome.