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.

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.

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.

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.

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.

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.

Non-Functioning Adrenocortical Carcinoma Presenting as Retroperitoneal Hemorrhage With Early Metastasis.

Adrenocortical cancer is a rare neoplasm with varied clinical presentation and overall poor outcome. This should be managed with timely intervention at highly specialized centers. Our aim is to report this rare case presentation of large non-functional adrenocortical cancer, complicated by spontaneous rupture while awaiting workup leading to life-threatening hemorrhage. Despite successful emergency radical surgical management and achieving negative margins, the patient developed early recurrence as intra-abdominal metastasis within two months. This can likely be attributed to the aggressive nature of the tumor as indicated by the high Ki-67 index or spillage of the tumor cells following spontaneous rupture. We recommend managing these non-functioning adrenocortical cancers as early as possible at highly specialized centers with reference to published standard guidelines.

Hepatomegaly, Elevated Hepatic Enzymes, and Bridging Fibrosis in Patients With Type 1 Diabetes Mellitus.

Glycogenic hepatopathy is a rare but reversible condition that includes acute liver dysfunction and hepatomegaly. This occurs due to excessive glycogen accumulation in the hepatocytes. It can occur in patients with poorly controlled type 1 diabetes mellitus. We are reporting a case of a 17-year-old girl who developed liver dysfunction following admission with diabetic ketoacidosis. Ultrasound abdomen confirmed hepatomegaly. However, with improvement in her metabolic control, her liver enzymes and hepatomegaly improved.

A Retrospective Study Assessing the Effect of Diabetes on Mortality in Patients With COVID-19 at a Teaching Hospital in the United Kingdom.

Aim The aim of the study was to compare the clinical characteristics and outcomes (mortality, intensive care admission, mechanical ventilation, and length of stay, LoS) of patients with and without diabetes with confirmed COVID-19.  Methods This retrospective study evaluated clinical and laboratory variables in adult inpatients from Brighton and Sussex University Hospitals NHS Trust with laboratory-confirmed COVID-19 between March 10, 2020, and June 30, 2020. Univariate and multivariate analyses were performed to compare the outcomes of patients with and without diabetes.  Results Over 457 patients were included in this study (140 with diabetes and 317 without diabetes), of which 143 (31.9%) died. The median age was 80 years and were predominantly males (59.1%). Baseline characteristics at the time of COVID-19 diagnosis demonstrated that the patients with diabetes were younger than those without diabetes (p=0.008). Mortality increased with age. There was no difference in adverse outcomes in those with and without diabetes. However, subgroup analysis of patients aged ≤60 years demonstrated a significantly increased mortality in those with diabetes (p=0.016). Patients with diabetes had an increased length-of-stay compared to those without diabetes, which was more evident in those aged ≤60 years. Conclusion Age is the most important predictor of mortality. Patients with diabetes did not have increased mortality from COVID-19, which is likely due to their younger age in our cohort. More patients with diabetes stayed in the hospital longer than seven days than those without diabetes.

A Case of Combined Diabetic Ketoacidosis and Hyperosmolar Hyperglycemic State in a Patient With COVID-19.

The novel coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2). Diabetes mellitus (DM) is one of the risk factors associated with severe illness in COVID-19 leading to increased hospital admissions and mortality. COVID-19 can precipitate hyperglycemic emergencies like diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS) in patients with DM. We present a case of a patient with COVID-19 admitted to the hospital with combined DKA and HHS. The case highlights the challenge of managing patients with DM suffering from COVID-19.

Reference genes for expression studies in hypoxia and hyperglycemia models in human umbilical vein endothelial cells.

Human umbilical vein endothelial cell (HUVEC)-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 hr either in euglycemic or in hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia for 1, 3, or 12 hr. Using whole-transcript arrays, we selected 10 commonly used reference genes with no significant expression variation across eight different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by qRT-PCR. These were ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-glucuronidase (GUSB), and ß-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 hr. Under hyperglycemia combined with hypoxia up to 12 hr, the expression of RPLP0, TFRC, GUSB, and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved.