Nicotinamide Adenine Dinucleotide Phosphate Oxidases in Glucose Homeostasis and Diabetes-Related Endothelial Cell Dysfunction.

Oxidative stress within the vascular endothelium, due to excess generation of reactive oxygen species (ROS), is thought to be fundamental to the initiation and progression of the cardiovascular complications of type 2 diabetes mellitus. The term ROS encompasses a variety of chemical species including superoxide anion (O2•-), hydroxyl radical (OH-) and hydrogen peroxide (H2O2). While constitutive generation of low concentrations of ROS are indispensable for normal cellular function, excess O2•- can result in irreversible tissue damage. Excess ROS generation is catalysed by xanthine oxidase, uncoupled nitric oxide synthases, the mitochondrial electron transport chain and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Amongst enzymatic sources of O2•- the Nox2 isoform of NADPH oxidase is thought to be critical to the oxidative stress found in type 2 diabetes mellitus. In contrast, the transcriptionally regulated Nox4 isoform, which generates H2O2, may fulfil a protective role and contribute to normal glucose homeostasis. This review describes the key roles of Nox2 and Nox4, as well as Nox1 and Nox5, in glucose homeostasis, endothelial function and oxidative stress, with a key focus on how they are regulated in health, and dysregulated in type 2 diabetes mellitus.

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.

Crosstalk of TLR4, vascular NADPH oxidase, and COVID-19 in diabetes: What are the potential implications?

Toll-like receptor 4 (TLR4) contributes to the pathophysiology of diabetes. This happens, at least in part, because TLR4 modulates the enzyme NADPH oxidase, a primary source of ROS in vascular structures. Increased oxidative stress disrupts key vascular signaling mechanisms and drives the progression of diabetes, elevating the likelihood of cardiovascular diseases. Recently, it has been shown that patients with diabetes are also at a higher risk of developing severe coronavirus disease 2019 (COVID-19). Given the importance of the interaction between TLR4 and NADPH oxidase to the disrupted diabetic vascular system, we put forward the hypothesis that TLR4-mediated NADPH oxidase-derived ROS might be a critical mechanism to help explain why this disparity appears in diabetic patients, but unfortunately, conclusive experimental evidence still lacks in the literature. Herein, we focus on discussing the pathological implications of this signaling communication in the diabetic vasculature and exploring this crosstalk in the context of diabetes-associated severe COVID-19.

Quercetin Attenuated Myeloperoxidase-Dependent HOCl Generation and Endothelial Dysfunction in Diabetic Vasculature.

Myeloperoxidase (MPO)-dependent hypochlorous acid (HOCl) generation plays crucial roles in diabetic vascular complications. As a natural polyphenol, quercetin has antioxidant properties in various diabetic models. Herein, we investigated the therapeutic mechanism for quercetin on MPO-mediated HOCl generation and endothelial dysfunction in diabetic vasculature. In vitro, the presence of MPO could amplify high glucose-induced endothelial dysfunction which was significantly inhibited by the NADPH oxidase inhibitor, HOCl or H2O2 scavengers, revealing the contribution of MPO/H2O2/HOCl to vascular endothelial injury. Furthermore, quercetin effectively inhibited MPO/high glucose-mediated HOCl generation and cytotoxicity to vascular endothelial cells. The inhibitive effect on MPO activity was related to the fact that quercetin reduced high glucose-induced H2O2 generation in endothelial cells and directly acted as a competitive substrate for MPO, thus limiting MPO/H2O2-dependent HOCl production. Moreover, quercetin could attenuate HOCl-caused endothelial dysfunction in endothelial cells and isolated aortas. In vivo, dietary quercetin significantly inhibited aortic endothelial dysfunction in diabetic mice, while this compound simultaneously suppressed vascular MPO expression and activity. Therefore, it was demonstrated herein that quercetin inhibited endothelial injury in diabetic vasculature via suppression of MPO/high glucose-dependent HOCl formation.

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.

Kanglexin accelerates diabetic wound healing by promoting angiogenesis via FGFR1/ERK signaling.

Diabetic foot is one of the main causes of non-traumatic amputation. However, there is still lack of effective drugs to treat diabetic foot in clinical practice. Kanglexin (KLX) is a new anthraquinone compound with cardiovascular protective effects. Here we report that KLX accelerates diabetic wound healing by promoting angiogenesis via FGFR1/ERK signaling. Firstly, KM mice were injected (ip) with streptozocin to establish type 1 diabetic model. The full thickness wound with the diameter of 5 mm was prepared on the back of each mice. The wounds were treated with KLX once a day for 14 consecutive days. Results showed that KLX significantly accelerated the closure of diabetic wounds. Pathological studies of skin tissues around the wounds showed that KLX promoted the formation of granulation tissue and new blood vessels, increased collagen deposition and reduced inflammatory cell infiltration. Besides, KLX significantly alleviated advanced glycation end products (AGEs) - induced abnormal proliferation, migration and tubule formation of human umbilical vein endothelial cells (HUVECs), and up-regulated phospho-ERK1/2 both in the diabetic wound tissue and AGEs - treated HUVECs. Moreover, molecular docking results indicated that KLX had the potential to bind with FGF receptor 1 (FGFR1), and subsequent experiments confirmed that FGFR1 inhibitor PD173074 reversed the effect of KLX on promoting the phosphorylation of ERK1/2 and angiogenesis, suggesting that KLX promoted angiogenesis through FGFR1/ERK signaling. In conclusion, our study provides a new effective compound for treating diabetic wounds. More importantly, KLX has the potential to be developed as a topical drug to promote diabetic wound healing.

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.

Impact of the acute local inhibition of soluble epoxide hydrolase on diabetic skin microcirculatory dysfunction.

The impact of the local inhibition of soluble epoxide hydrolase, which metabolizes vasodilator and anti-inflammatory epoxyeicosanoids, on diabetic skin microvascular dysfunction was assessed. In diabetic db/db mice, basal skin blood flow assessed using laser Doppler imaging was similar to that of control mice, but thermal hyperemia was markedly reduced. At 2 h after the topical administration of an aqueous gel containing the soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB: 400 mg/L), the peak concentration of t-AUCB was detected in the skin of diabetic mice, which quickly decreased thereafter. In parallel, 2 h after application of t-AUCB treatment, thermal hyperemia was increased compared to the control gel. Quantification of t-AUCB in plasma of treated animals showed no or low systemic diffusion. Furthermore, haematoxylin and eosin histological staining of skin biopsies showed that skin integrity was preserved in t-AUCB-treated mice. Finally, for pig ear skin, a surrogate for human skin, using Franz diffusion cells, we observed a continuous diffusion of t-AUCB from 2 h after application to beyond 24 h. A single topical administration of a soluble epoxide hydrolase inhibitor improves microcirculatory function in the skin of db/db mice and might represent a new therapeutic approach for preventing the development of skin complications in diabetic patients.

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.

Augmented contractility of murine femoral arteries in a streptozotocin diabetes model is related to increased phosphorylation of MYPT1.

Diabetes mellitus (DM) is a metabolic disorder with high prevalence, and a major risk factor for macro- and microvascular abnormalities. This study was undertaken to explore the mechanisms of hypercontractility of murine femoral arteries (FA) obtained from mice with streptozotocin (STZ)-induced diabetes and its relation to the phosphorylation profile of the myosin phosphatase target subunit 1, MYPT1. The immunoreactivity of MYPT1 toward phospho-MYPT1-T696, MYPT1-T853, or MYPT1-S695, used as a read out for MYPT1 phosphorylation, has been studied by Western Blotting. Contractile activity of FA from control and STZ mice has been studied by wire myography. At basal conditions (no treatment), the immunoreactivity of MYPT1-T696/T853 was ~2-fold higher in the STZ arteries compared with controls. No changes in MYPT1-T696/853 phosphorylation were observed after stimulation with the Thromboxan-A2 analog, U46619. Neither basal nor U46619-stimulated phosphorylation of MYPT1 at S695 was affected by STZ treatment. Mechanical distensibility and basal tone of FA obtained from STZ animals were similar to controls. Maximal force after treatment of FA with the contractile agonists phenylephrine (10 µmol/L) or U46619 (1 µmol/L) was augmented in the arteries of STZ mice by ~2- and ~1.5-fold, respectively. In summary, our study suggests that development of a hypercontractile phenotype in murine FA in STZ diabetes is at least partially related to an increase in phosphorylation of MLCP at MYPT1-T696/853. Interestingly, the phosphorylation at S695 site was not altered in STZ-induced diabetes, supporting the view that S695 may serve as a sensor for mechanical activity which is not directly involved in tone regulation.

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.

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.

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.

FAL1 regulates endothelial cell proliferation in diabetic arteriosclerosis through PTEN/AKT pathway.

OBJECTIVE: This study aims to investigate the role of FAL1 in the occurrence and progression of diabetic arteriosclerosis and its underlying mechanism. PATIENTS AND METHODS: FAL1 expression in coronary artery disease (CAD) tissues, normal artery tissues, and tumor necrosis factor-α (TNF-α)-induced endothelial cells was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The regulatory effects of FAL1 on cell proliferation, migration, and cell cycle were examined by cell counting kit-8 (CCK-8) assay, transwell assay, and flow cytometry, respectively. Western blot was used to detect protein expressions of proliferation-related gene PCNA (proliferating cell nuclear antigen), cell cycle-related genes cyclin D1, PTEN (phosphatase and tensin homolog deleted on chromosome ten) and AKT (protein kinase B) in HUVECs. Subsequently, rescue experiments were performed to assess whether PTEN/AKT signaling pathway is activated during the process of FAL1-regulated proliferation and migration of HUVECs. RESULTS: FAL1 was highly expressed in CAD tissues and TNF-α-induced endothelial cells compared with that of controls. Overexpression of FAL1 in HUVECs promoted cell cycle, proliferation, and migration. FAL1 activated PTEN/AKT pathway in HUVECs, which was partially reversed by PTEN overexpression. CONCLUSIONS: Highly expressed FAL1 can promote proliferation and migration of endothelial cells through activating PTEN/AKT signaling pathway.

Comprehensive insight into functional interaction between GNB3 C825T and eNOS T-786C, G894T gene polymorphisms and association with susceptibility to diabetic erectile dysfunction.

BACKGROUND: No study has assessed the possible involvement of endothelial nitric oxide synthase (eNOS) T-786C and G894T and G-protein ß3 subunit (GNB3) C825T polymorphisms with susceptibility to diabetic vasculogenic erectile dysfunction (VED) in North African subjects. OBJECTIVES: Our aim was to evaluate the interaction and association between these gene polymorphisms and this disorder. MATERIALS AND METHODS: A total of 164 type 2 diabetes patients with VED diagnosed with penile color Doppler ultrasonography and 148 age-matched healthy volunteers were genotyped for the rs1799983 (G894T) and rs2070744 (T-786C) of the eNOS gene and the rs5443 (C825T) of the GNB3 gene using the PCR-RFLP method. RESULTS: A significant association of the eNOS G894T (p = 0.005) and T-786C (p = 0.02) with altered susceptibility to VED was observed. The risk also holds for the G894T and T-786C eNOS gene polymorphisms when excluding patients with dyslipidemia and cardiovascular diseases (p = 1.7·10-4 and p = 3.2·10-5 , respectively). The univariate odds ratio associated with CC alleles of the eNOS T-786C revealed a four times increased risk for VED (OR = 4.04; 95% CI = 1.53-10.67; p = 0.006). VED risk was also associated with the G894T variant under dominant model (p = 0.002) and the T-786C variant under recessive model (p = 0.004). Furthermore, the concomitant presence of the combined genotypes of the 894T and 786T strongly affected the predisposition to VED (p = 0.007). DISCUSSION AND CONCLUSION: Our study gave a comprehensive insight into functional interaction between GNB3 and eNOS gene polymorphisms and suggests that the eNOS G894T and T-786C variants are strong predisposing factors of VED susceptibility within men with type 2 diabetes.

Vascular endothelial growth factor over-expressed mesenchymal stem cells-conditioned media ameliorate palmitate-induced diabetic endothelial dysfunction through PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathway.

In the pathogenesis of diabetes mellitus (DM), islet microvasculares are severely damaged due to glucolipotoxicity and other reasons. Vascular endothelial growth factor (VEGF) is an indispensable and specific angiogenic factor in the pathogenesis and treatment of diabetic islet microvascular disease. Mesenchymal stem cells (MSCs) are regarded as a promising treatment of diabetes because of their immunosuppressive effect and multipotential differentiation potency. In this study, we tested whether MSCs over-expressing VEGF conditioned medium (MSC-VEGF-CM) could ameliorate pancreatic islet endothelial cells (MS-1) dysfunction induced by a common diabetic inducer palmitate (PA). We found that cell survival and migration were restrained by PA and partly repaired by the pro-protected of MSC-VEGF-CM. Meanwhile, PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathways were also up-regulated. Though apoptosis-related proteins, caspase-3 and caspase-9, had no significantly suppressed between MSC-VEGF-CM and MSC-CM alone, the expression levels of vascular surface factors such as CD31, VE-cadherin, occludin and ICAM-1, were remarkably up-regulated by the pro-protected of MSC-VEGF-CM. Our data suggested that MSC-VEGF-CM had therapeutic effect on the PA-induced dysfunction through the re-activation of PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathways.

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.

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.

Acid Sphingomyelinase Down-regulation Alleviates Vascular Endothelial Insulin Resistance in Diabetic Rats.

Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in patients with type 2 diabetes. Acid sphingomyelinase (ASM) is a soluble glycoprotein which plays a vital role in the development and progression of various diseases such as cardiovascular and metabolic diseases. However, it remains unknown if ASM regulates insulin resistance in vascular endothelial cells in type 2 diabetes. ASM down-regulation with gene silencing and selective inhibitor amitriptyline was used in the rat aortic endothelial cells (RAECs) treated with palmitic acid (PA), a common saturated free fatty acid, which is thought to be the major cause of insulin resistance. It was shown that ASM down-regulation increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of pIRS-1-ser307 and AKT-ser473 via ceramide, consequently resulting in the decrease of the production of endothelial nitric oxide synthase (eNOS) and nitric oxide in PA-induced RAECs. We further found that ASM down-regulation blocked the Nox2- and Nox4-dependent superoxide (O2 -· ) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vivo, amitriptyline relieved the vasodilatory response to acetylcholine and restored the level of ceramide, Nox2 and Nox4 in the aorta endothelium of high-fat diet-fed rats following an injection of streptozotocin. Taken together, these results suggest that ASM down-regulation can improve endothelial insulin resistance which is attributed to inhibiting redox signalling in RAECs. Thus, these data support the idea that ASM is a promising clinical biomarker and potential therapeutic target for diabetic vascular complication.

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.