Human blood vessel organoids as a model of diabetic vasculopathy.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

Diabetes Impaired Ischemia-Induced PDGF (Platelet-Derived Growth Factor) Signaling Actions and Vessel Formation Through the Activation of Scr Homology 2-Containing Phosphatase-1.

Objective: Critical limb ischemia is a major complication of diabetes characterized by insufficient collateral vessel development and proper growth factor signaling unresponsiveness. Although mainly deactivated by hypoxia, phosphatases are important players in the deregulation of proangiogenetic pathways. Previously, SHP-1 (Scr homology 2-containing phosphatase-1) was found to be associated with the downregulation of growth factor actions in the diabetic muscle. Thus, we aimed to gain further understanding of the impact of SHP-1 on smooth muscle cell (SMC) function under hypoxic and diabetic conditions. Approach and Results: Despite being inactivated under hypoxic conditions, high glucose level exposure sustained SHP-1 phosphatase activity in SMC and increased its interaction with PDGFR (platelet-derived growth factor receptor)-ß, thus reducing PDGF proangiogenic actions. Overexpression of an inactive form of SHP-1 fully restored PDGF-induced proliferation, migration, and signaling pathways in SMC exposed to high glucose and hypoxia. Nondiabetic and diabetic mice with deletion of SHP-1 specifically in SMC were generated. Ligation of the femoral artery was performed, and blood flow was measured for 4 weeks. Blood flow reperfusion, vascular density and maturation, and limb survival were all improved while vascular apoptosis was attenuated in diabetic SMC-specific SHP-1 null mice as compared to diabetic mice. Conclusions: Diabetes and high glucose level exposure maintained SHP-1 activity preventing hypoxia-induced PDGF actions in SMC. Specific deletion of SHP-1 in SMC partially restored blood flow reperfusion in the diabetic ischemic limb. Therefore, local modulation of SHP-1 activity in SMC could represent a potential therapeutic avenue to improve the proangiogenic properties of SMC under ischemia and diabetes.

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes.

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1-/- mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.

CEACAM1 Inhibited IκB-α/NF-κB Signal Pathway Via Targeting MMP-9/TIMP-1 Axis in Diabetic Atherosclerosis.

Atherosclerosis (AS) is the most common and serious complication in type 2 diabetes mellitus (T2DM). Recent studies have emphasized that inflammation is the main cause of atherosclerosis. Studies have shown that carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) regulates the expression of matrix metallopeptidase 9 (MMP-9) after ischemic stroke to reduce inflammation. The aim of this study was to elucidate potential molecular mechanism of CEACAM1 on the inflammatory response in atherosclerosis. The serum levels of CEACAM1, MMP-9, and tissue inhibitors of metalloproteinase 1 (TIMP-1) in T2DM patients and healthy control was detected. The results showed that the levels of CEACAM1 and TIMP-1 were significantly decreased, and the levels of MMP-9 were significantly higher than those in the control group. Moreover, we also observed the effect of CEACAM1 on atherosclerosis in T2DM rats. Hematoxylin & eosin (HE) staining and oil red staining showed that CEACAM1 recombinant protein reduced intima-media thickness and the area of atherosclerotic plaques. To further explore the molecular mechanism of CEACAM1 regulating MMP-9/TIMP-1, we conducted experiments in rat aorta vascular endothelial cells and rat aorta smooth muscle cells. The result showed that CEACAM1 inhibits inflammatory response via MMP-9/TIMP-1 axis. Taken together, CEACAM1 attenuates diabetic atherosclerosis by inhibition of IκB/NF-κB signal pathway via MMP-9/TIMP-1 axis, which indicate that CEACAM1 is potentially amenable to therapeutic manipulation for clinical application in atherosclerosis in T2DM.

SIRT1 activation promotes angiogenesis in diabetic wounds by protecting endothelial cells against oxidative stress.

OBJECTIVE: Chronic wounds are a devastating complication of diabetes and can lead to amputations or even death. Current medical therapies are insufficient to accelerate its repair. The objective of this study was to explore the role of Sirtuin1 (SIRT1) in diabetic wounds. METHODS AND MATERIALS: Perilesional skin tissue samples from diabetic ulcers and normoglycemic trauma wounds were used to detect SIRT1 expression and oxidative stress levels. In a diabetic mouse model, SIRT1 was pharmacologically activated to attenuate angiogenesis and accelerate wound closure. Finally, in vitro experiments were performed to elucidate some of the mechanisms by which SIRT1 activation promotes angiogenesis in diabetic wound healing. RESULTS: We found that skin tissue from diabetes patients showed lower expression of SIRT1 and severe oxidative stress. Decreased SIRT1 expression was observed in skin tissue from streptozocin (STZ)-induced diabetic mice and was associated with impaired wound healing. In addition, the wounds of STZ-induced diabetic mice treated with SRT1720 (a specific SIRT1 activator) demonstrated locally improved wound healing and angiogenesis. In the in vitro experiment, similar results were observed. Under hyperglycemia conditions, human umbilical vein endothelial cells (HUVECs) showed lower expression of SIRT1 and higher levels of reactive oxygen species (ROS) production. Furthermore, the migration, proliferation and in vitro tube formation ability of HUVECs were impaired under hyperglycemia conditions, and SRT1720 treatment rescued these impairments and decreased ROS production in HUVECs. CONCLUSIONS: This study provides experimental evidence that SIRT1 activation could improve angiogenesis in wounds in vitro and in vivo and that sirtuin1 activation accelerates wound healing in diabetic mice by promoting angiogenesis. These positive therapeutic effects may be mediated by protecting vascular endothelial cells from oxidative stress injury. This study suggested that SIRT1 may serve as a potentially important and potent therapeutic target for treating diabetic ulcers.

Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis.

Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1+/+ ; Ldlr-/- (single knock-out) and Ddr1-/- ; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.

Endothelial Glycocalyx as a Shield Against Diabetic Vascular Complications: Involvement of Hyaluronan and Hyaluronidases.

The endothelial glycocalyx (EG), which covers the apical surface of the endothelial cells and floats into the lumen of the vessels, is a key player in vascular integrity and cardiovascular homeostasis. The EG is composed of PGs (proteoglycans), glycoproteins, glycolipids, and glycosaminoglycans, in particular hyaluronan (HA). HA seems to be implicated in most of the functions described for EG such as creating a space between blood and the endothelium, controlling vessel permeability, restricting leukocyte and platelet adhesion, and allowing an appropriate endothelial response to flow variation through mechanosensing. The amount of HA in the EG may be regulated by HYAL (hyaluronidase) 1, the most active somatic hyaluronidase. HYAL1 seems enriched in endothelial cells through endocytosis from the bloodstream. The role of the other main somatic hyaluronidase, HYAL2, in the EG is uncertain. Damage to the EG, accompanied by shedding of one or more of its components, is an early sign of various pathologies including diabetes mellitus. Shedding increases the blood or plasma concentration of several EG components, such as HA, heparan sulfate, and syndecan. The plasma levels of these molecules can then be used as sensitive markers of EG degradation. This has been shown in type 1 and type 2 diabetic patients. Recent experimental studies suggest that preserving the size and amount of EG HA in the face of diabetic insults could be a useful novel therapeutic strategy to slow diabetic complications. One way to achieve this goal, as suggested by a murine model of HYAL1 deficiency, may be to inhibit the function of HYAL1. The same approach may succeed in other pathological situations involving endothelial dysfunction and EG damage.

Akt2 (Protein Kinase B Beta) Stabilizes ATP7A, a Copper Transporter for Extracellular Superoxide Dismutase, in Vascular Smooth Muscle: Novel Mechanism to Limit Endothelial Dysfunction in Type 2 Diabetes Mellitus.

OBJECTIVE: Copper transporter ATP7A (copper-transporting/ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM (type2 diabetes mellitus) mice and human in which insulin-Akt (protein kinase B) pathway is selectively impaired. APPROACH AND RESULTS: Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet-induced or db/db T2DM mice. Akt2 (protein kinase B beta) activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B-/- (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhance insulin-Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2-/- vessels or VSMCs, which is rescued by ATP7A overexpression. CONCLUSION: Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.

Association of serum proprotein convertase subtilisin/kexin type 9 with early atherosclerosis in newly diagnosed type 2 diabetes mellitus.

BACKGROUND AND AIM: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is rapidly gaining attention as a potential risk of developing atherosclerosis due to its crucial role in the regulation of low-density lipoprotein cholesterol (LDL-C) metabolism. The present study investigated the relationship between serum PCSK9 levels and early atherosclerosis as assessed by carotid intimal-medial thickness (CIMT) and brachial-ankle pulse wave velocity (ba-PWV) in newly diagnosed type 2 diabetes mellitus (T2DM). METHODS AND RESULTS: A total of 100 newly diagnosed T2DM were enrolled and further divided into the thickened CIMT group (n = 41) and the non-thickened CIMT group (n = 59) according to the results of color Doppler ultrasonography. Serum PCSK9 levels, CIMT, ba-PWV, and metabolic parameters were measured. Patients in the thickened CIMT group had higher serum PCSK9 levels than patients in the non-thickened CIMT group (all P < 0.05). CIMT and ba-PWV were both positively correlated to serum PCSK9 levels, while serum PCSK9 levels were positively correlated to white blood cell count, neutrophil, lymphocyte, and high-sensitivity C-reactive protein (P < 0.05). Multiple linear regression indicated that serum PCSK9 level was an independent predictor of CIMT (ß = 0.637, P < 0.001) and ba-PWV (ß = 0.600, P < 0.001). Binary logistic regression analysis showed that serum PCSK9 levels were independent risk factors of thickened CIMT [OR = 1.120, 95%CI (1.041-1.204), P = 0.002]. CONCLUSION: Serum PCSK9 levels are significantly correlated with CIMT and ba-PWV, independent of CAD risk factors. Therefore, serum PCSK9 level may have the potential to serve as a prescriptive biomarker for early arteriosclerosis in newly diagnosed T2DM.

Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice.

Coronary microvascular rarefaction, due to endothelial cell (EC) dysfunction, is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial function. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca2+ concentration ([Ca2+]mito) compared with the control. Among several regulatory proteins for [Ca2+]mito, hexokinase 1 (HK1) and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from nondiabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in MCECs. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca2+]mito, but also reduced mitochondrial reactive oxygen species production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.

The role of dipeptidylpeptidase-4 inhibitors in management of cardiovascular disease in diabetes; focus on linagliptin.

Multiple population based analyses have demonstrated a high incidence of cardiovascular disease (CVD) and cardiovascular (CV) mortality in subjects with T2DM that reduces life expectancy by as much as 15 years. Importantly, the CV system is particularly sensitive to the metabolic and immune derangements present in obese pre-diabetic and diabetic individuals; consequently, CV dysfunction is often the initial CV derangement to occur and promotes the progression to end organ/tissue damage in T2DM. Specifically, diabetic CVD can manifest as microvascular complications, such as nephropathy, retinopathy, and neuropathy, as well as, macrovascular impairments, including ischemic heart disease, peripheral vascular disease, and cerebrovascular disease. Despite some progress in prevention and treatment of CVD, mainly via blood pressure and dyslipidemia control strategies, the impact of metabolic disease on CV outcomes is still a major challenge and persists in proportion to the epidemics of obesity and diabetes. There is abundant pre-clinical and clinical evidence implicating the DPP-4-incretin axis in CVD. In this regard, linagliptin is a unique DPP-4 inhibitor with both CV and renal safety profiles. Moreover, it exerts beneficial CV effects beyond glycemic control and beyond class effects. Linagliptin is protective for both macrovascular and microvascular complications of diabetes in preclinical models, as well as clinical models. Given the role of endothelial-immune cell interactions as one of the key events in the initiation and progression of CVD, linagliptin modulates these cell-cell interactions by affecting two important pathways involving stimulation of NO signaling and potent inhibition of a key immunoregulatory molecule.

p66Shc gene expression in peripheral blood mononuclear cells and progression of diabetic complications.

BACKGROUND: The risk of diabetic complications is modified by genetic and epigenetic factors. p66Shc drives the hyperglycaemic cell damage and its deletion prevents experimental diabetic complications. We herein tested whether p66Shc expression in peripheral blood mononuclear cells (PBMCs) predicts adverse outcomes in people with diabetes. METHODS: In a cohort of 100 patients with diabetes (16 type 1 and 84 type 2), we quantified baseline p66Shc expression in PBMCs by quantitative PCR. Patients were extensively characterized for demographics, anthropometrics, biochemical data, prevalence of complications, and medications. With a pseudo-prospective design, we retrieved cardiovascular death, major adverse cardiovascular events (MACE), and new occurrence of micro- or macroangiopathy during follow-up. RESULTS: At baseline, patients were on average 60 year old, with 10-year diabetes duration, and overall poor glycaemic control (HbA1c 7.8%). Patients with high versus low p66Shc expression (based on median value) had very similar baseline characteristics. Average p66Shc expression did not differ by presence/absence of complications. During a median 5.6-year follow-up, the primary endpoint of cardiovascular death or MACE occurred in 22 patients, but no relation was detected between cardiovascular outcomes and p66Shc expression. In patients who developed new complications at follow-up, baseline p66Shc was significantly higher, especially for macroangiopathy. The incidence of new macroangiopathy was > 3-times higher in patients with high versus those with low baseline p66Shc expression. CONCLUSIONS: p66Shc expression in PBMCs was not associated with prevalent diabetic complications but predicted new onset of complications, especially macroangiopathy, although no relation with hard cardiovascular endpoints was detected.

Resveratrol Mitigates High-Fat Diet-Induced Vascular Dysfunction by Activating the Akt/eNOS/NO and Sirt1/ER Pathway.

We investigated whether resveratrol (RSV) can attenuate obesity and diabetes progression and improve diabetes-induced vascular dysfunction, and we attempted to delineate its underlying mechanisms. Male C57Bl/6 mice were administered a high-fat diet (HFD) for 17 weeks. Mice developed type 2 diabetes with increased body weight, hyperglycemia, hyperinsulinemia, and hyperlipidemia. Oral gavage with RSV significantly reversed the symptoms induced by the HFD. Insulin sensitivity likewise improved after the RSV intervention in these mice. Phenylephrine-induced cremaster arteriolar constriction was impaired, whereas RSV treatment significantly mitigated the vessel responsiveness to phenylephrine. The obese diabetic mice exhibited increased leukocyte rolling, adhesion, and transmigration in the postcapillary venules of the cremaster muscle. By contrast, RSV treatment significantly attenuated HFD-induced extravasation. RSV significantly recovered phosphorylated Akt and eNOS expression in the thoracic aorta. In addition, activated adenosine monophosphate-activated protein kinase in the thoracic aorta was involved in the improvement of epithelial function after RSV intervention. RSV considerably upregulated the plasma NO level in HFD mice. Moreover, RSV-enhanced human umbilical vein endothelial cells healing through Sirt1/ER pathway may be involved in the prevention of leukocyte extravasation. Collectively, RSV attenuates diabetes-induced vascular dysfunction by activating Akt/eNOS/NO and Sirt1/ER pathway. Our mechanistic study provides a potential RSV-based therapeutic strategy against cardiovascular disease.

Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.

Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H2O2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H2O2-MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases.

Regulation of high glucose-induced apoptosis of brain pericytes by mitochondrial CA VA: A specific target for prevention of diabetic cerebrovascular pathology.

Events responsible for cerebrovascular disease in diabetes are not fully understood. Pericyte loss is an early event that leads to endothelial cell death, microaneurysms, and cognitive impairment. A biochemical mechanism underlying pericyte loss is rapid respiration (oxidative metabolism of glucose). This escalation in respiration results from free influx of glucose into insulin-insensitive tissues in the face of high glucose levels in the blood. Rapid respiration generates superoxide, the precursor to all reactive oxygen species (ROS), and results in pericyte death. Respiration is regulated by carbonic anhydrases (CAs) VA and VB, the two isozymes expressed in mitochondria, and their pharmacologic inhibition with topiramate reduces respiration, ROS, and pericyte death. Topiramate inhibits both isozymes; therefore, in the earlier studies, their individual roles were not discerned. In a recent genetic study, we showed that mitochondrial CA VA plays a significant role in regulation of reactive oxygen species and pericyte death. The role of CA VB was not addressed. In this report, genetic knockdown and overexpression studies confirm that mitochondrial CA VA regulates respiration in pericytes, whereas mitochondrial CA VB does not contribute significantly. Identification of mitochondrial CA VA as a sole regulator of respiration provides a specific target to develop new drugs with fewer side effects that may be better tolerated and can protect the brain from diabetic injury. Since similar events occur in the capillary beds of other insulin-insensitive tissues such as the eye and kidney, these drugs may also slow the onset and progression of diabetic disease in these tissues.

A randomised, active- and placebo-controlled, three-period crossover trial to investigate short-term effects of the dipeptidyl peptidase-4 inhibitor linagliptin on macro- and microvascular endothelial function in type 2 diabetes.

BACKGROUND: Studies of dipeptidyl peptidase (DPP)-4 inhibitors report heterogeneous effects on endothelial function in patients with type 2 diabetes (T2D). This study assessed the effects of the DPP-4 inhibitor linagliptin versus the sulphonylurea glimepiride and placebo on measures of macro- and microvascular endothelial function in patients with T2D who represented a primary cardiovascular disease prevention population. METHODS: This crossover study randomised T2D patients (n = 42) with glycated haemoglobin (HbA1c) ≤7.5%, no diagnosed macro- or microvascular disease and on stable metformin background to linagliptin 5 mg qd, glimepiride 1-4 mg qd or placebo for 28 days. Fasting and postprandial macrovascular endothelial function, measured using brachial flow-mediated vasodilation, and microvascular function, measured using laser-Doppler on the dorsal thenar site of the right hand, were analysed after 28 days. RESULTS: Baseline mean (standard deviation) age, body mass index and HbA1c were 60.3 (6.0) years, 30.3 (3.0) kg/m2 and 7.41 (0.61)%, respectively. After 28 days, changes in fasting flow-mediated vasodilation were similar between the three study arms (treatment ratio, gMean [90% confidence interval]: linagliptin vs glimepiride, 0.884 [0.633-1.235]; linagliptin vs placebo, 0.884 [0.632-1.235]; glimepiride vs placebo, 1.000 [0.715-1.397]; P = not significant for all comparisons). Similarly, no differences were seen in postprandial flow-mediated vasodilation. However, under fasting conditions, linagliptin significantly improved microvascular function as shown by a 34% increase in hyperaemia area (P = 0.045 vs glimepiride), a 34% increase in resting blow flow (P = 0.011 vs glimepiride, P = 0.003 vs placebo), and a 25% increase in peak blood flow (P = 0.009 vs glimepiride, P = 0.003 vs placebo). There were no significant differences between treatments in postprandial changes. Linagliptin had no effect on heart rate or blood pressure. Rates of overall adverse events with linagliptin, glimepiride and placebo were 27.5, 61.0 and 35.0%, respectively. Fewer hypoglycaemic events were seen with linagliptin (5.0%) and placebo (2.5%) than with glimepiride (39.0%). CONCLUSIONS: Linagliptin had no effect on macrovascular function in T2D, but significantly improved microvascular function in the fasting state. Trial registration ClinicalTrials.gov identifier-NCT01703286; registered October 1, 2012.

Poly-ADP-ribose polymerase inhibition enhances ischemic and diabetic wound healing by promoting angiogenesis.

OBJECTIVE: Chronic nonhealing wounds are a major health problem for patients in the United States and worldwide. Diabetes and ischemia are two major risk factors behind impaired healing of chronic lower extremity wounds. Poly-ADP-ribose polymerase (PARP) is found to be overactivated with both ischemic and diabetic conditions. This study seeks a better understanding of the role of PARP in ischemic and diabetic wound healing, with a specific focus on angiogenesis and vasculogenesis. METHODS: Ischemic and diabetic wounds were created in FVB/NJ mice and an in vitro scratch wound model. PARP inhibitor PJ34 was delivered to the animals at 10 mg/kg/d through implanted osmotic pumps or added to the culture medium, respectively. Animal wound healing was assessed by daily digital photographs. Animal wound tissues, peripheral blood, and bone marrow cells were collected at different time points for further analysis with Western blot and flow cytometry. Scratch wound migration and invasion angiogenesis assays were performed using human umbilical vein endothelial cells (HUVECs). Measurements were reported as mean ± standard deviation. Continuous measurements were compared by t-test. P < .05 was considered statistically significant. RESULTS: A significant increase in PARP activity was observed under ischemic and diabetic conditions that correlated with delayed wound healing and slower HUVEC migration. The beneficial effect of PARP inhibition with PJ34 on ischemic and diabetic wound healing was observed in both animal and in vitro models. In the animal model, the percentage of wound healing was significantly enhanced from 43% ± 6% to 71% ± 9% (P < .05) by day 7 with the addition of PJ34. PARP inhibition promoted angiogenesis at the ischemic and diabetic wound beds as evidenced by significantly higher levels of endothelial cell markers (vascular endothelial growth factor receptor 2 [VEGFR2] and endothelial nitric oxide synthase) in mice treated with PJ34 compared with controls. Flow cytometry analysis of peripheral blood mononuclear cells showed that PARP inhibition increased mobilization of endothelial progenitor cells (VEGFR2+/CD133+ and VEGFR2+/CD34+) into the systemic circulation. Furthermore, under in vitro hyperglycemia and hypoxia conditions, PARP inhibition enhanced HUVEC migration and invasion in Boyden chamber assays by 80% and 180% (P < .05), respectively. CONCLUSIONS: Delayed healing in ischemic and diabetic wounds is caused by PARP hyperactivity, and PARP inhibition significantly enhanced ischemic and diabetic wound healing by promoting angiogenesis.

P66Shc-Induced MicroRNA-34a Causes Diabetic Endothelial Dysfunction by Downregulating Sirtuin1.

OBJECTIVE: Diabetes mellitus causes vascular endothelial dysfunction and alters vascular microRNA expression. We investigated whether endothelial microRNA-34a (miR-34a) leads to diabetic vascular dysfunction by targeting endothelial sirtuin1 (Sirt1) and asked whether the oxidative stress protein p66Shc governs miR-34a expression in the diabetic endothelium. APPROACH AND RESULTS: MiR-34a is upregulated, and Sirt1 downregulated, in aortic endothelium of db/db and streptozotocin-induced diabetic mice. Systemic administration of miR-34a inhibitor, or endothelium-specific knockout of miR-34a, prevents downregulation of aortic Sirt1 and rescues impaired endothelium-dependent aortic vasorelaxation induced by diabetes mellitus. Moreover, overexpression of Sirt1 mitigates impaired endothelium-dependent vasorelaxation caused by miR-34a mimic ex vivo. Systemic infusion of miR-34a inhibitor or genetic ablation of endothelial miR-34a prevents downregulation of endothelial Sirt1 by high glucose. MiR-34a is upregulated, Sirt1 is downregulated, and oxidative stress (hydrogen peroxide) is induced in endothelial cells incubated with high glucose or the free fatty acid palmitate in vitro. Increase of hydrogen peroxide and induction of endothelial miR-34a by high glucose or palmitate in vitro is suppressed by knockdown of p66shc. In addition, overexpression of wild-type but not redox-deficient p66Shc upregulates miR-34a in endothelial cells. P66Shc-stimulated upregulation of endothelial miR-34a is suppressed by cell-permeable antioxidants. Finally, mice with global knockdown of p66Shc are protected from diabetes mellitus-induced upregulation of miR-34a and downregulation of Sirt1 in the endothelium. CONCLUSIONS: These data show that hyperglycemia and elevated free fatty acids in the diabetic milieu recruit p66Shc to upregulate endothelial miR-34a via an oxidant-sensitive mechanism, which leads to endothelial dysfunction by targeting Sirt1.

Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2.