Microvascular Dysfunction In Diabetes Research Articles

Circulating exosomal mir-16-2-3p is associated with coronary microvascular dysfunction in diabetes through regulating the fatty acid degradation of endothelial cells

BackgroundCoronary microvascular dysfunction (CMD) is a frequent complication of diabetes mellitus (DM) characterized by challenges in both diagnosis and intervention. Circulating levels of microRNAs are increasingly recognized as potential biomarkers for cardiovascular diseases.MethodsSerum exosomes from patients with DM, DM with coronary microvascular dysfunction (DM-CMD) or DM with coronary artery disease (DM-CAD) were extracted for miRNA sequencing. The expression of miR-16-2-3p was assessed in high glucose-treated human aortic endothelial cells and human cardiac microvascular endothelial cells. Fluorescence in situ hybridization (FISH) was used to detect miR-16-2-3p within the myocardium of db/db mice. Intramyocardial injection of lentivirus overexpressing miR-16-2-3p was used to explore the function of the resulting gene in vivo. Bioinformatic analysis and in vitro assays were carried out to explore the downstream function and mechanism of miR-16-2-3p. Wound healing and tube formation assays were used to explore the effect of miR-16-2-3p on endothelial cell function.ResultsmiR-16-2-3p was upregulated in circulating exosomes from DM-CMD, high glucose-treated human cardiac microvascular endothelial cells and the hearts of db/db mice. Cardiac miR-16-2-3p overexpression improved cardiac systolic and diastolic function and coronary microvascular reperfusion. In vitro experiments revealed that miR-16-2-3p could regulate fatty acid degradation in endothelial cells, and ACADM was identified as a potential downstream target. MiR-16-2-3p increased cell migration and tube formation in microvascular endothelial cells.ConclusionsOur findings suggest that circulating miR-16-2-3p may serve as a biomarker for individuals with DM-CMD. Additionally, miR-16-2-3p appears to alleviate coronary microvascular dysfunction in diabetes by modulating ACADM-mediated fatty acid degradation in endothelial cells.

Diabetes and the Microvasculature of the Bone and Marrow.

The purpose of this review is to highlight the evidence of microvascular dysfunction in bone and marrow and its relation to poor skeletal outcomes in diabetes mellitus. Diabetes mellitus is characterized by chronic hyperglycemia, which may lead to microangiopathy and macroangiopathy. Micro- and macroangiopathy have been diagnosed in Type 1 and Type 2 diabetes, coinciding with osteopenia, osteoporosis, enhanced fracture risk and delayed fracture healing. Microangiopathy has been reported in the skeleton, correlating with reduced blood flow and perfusion, vasomotor dysfunction, microvascular rarefaction, reduced angiogenic capabilities, and augmented vascular permeability. Microangiopathy within the skeleton may be detrimental to bone and manifest as, among other clinical abnormalities, reduced mass, enhanced fracture risk, and delayed fracture healing. More investigations are required to elucidate the various mechanisms by which diabetic microvascular dysfunction impacts the skeleton.

Early left ventricular microvascular dysfunction in diabetic pigs: a longitudinal quantitative myocardial perfusion CMR study

BackgroundMicrovascular pathology is one of the main characteristics of diabetic cardiomyopathy; however, the early longitudinal course of diabetic microvascular dysfunction remains uncertain. This study aimed to investigate the early dynamic changes in left ventricular (LV) microvascular function in diabetic pig model using the cardiac magnetic resonance (CMR)-derived quantitative perfusion technique.MethodsTwelve pigs with streptozotocin-induced diabetes mellitus (DM) were included in this study, and longitudinal CMR scanning was performed before and 2, 6, 10, and 16 months after diabetic modeling. CMR-derived semiquantitative parameters (upslope, maximal signal intensity, perfusion index, and myocardial perfusion reserve index [MPRI]) and fully quantitative perfusion parameters (myocardial blood flow [MBF] and myocardial perfusion reserve [MPR]) were analyzed to evaluate longitudinal changes in LV myocardial microvascular function. Pearson correlation was used to analyze the relationship between LV structure and function and myocardial perfusion function.ResultsWith the progression of DM duration, the upslope at rest showed a gradually increasing trend (P = 0.029); however, the upslope at stress and MBF did not change significantly (P > 0.05). Regarding perfusion reserve function, both MPRI and MPR showed a decreasing trend with the progression of disease duration (MPRI, P = 0.001; MPR, P = 0.042), with high consistency (r = 0.551, P < 0.001). Furthermore, LV MPR is moderately associated with LV longitudinal strain (r = − 0.353, P = 0.022), LV remodeling index (r = − 0.312, P = 0.033), fasting blood glucose (r = − 0.313, P = 0.043), and HbA1c (r = − 0.309, P = 0.046). Microscopically, pathological results showed that collagen volume fraction increased gradually, whereas no significant decrease in microvascular density was observed with the progression of DM duration.ConclusionsMyocardial microvascular reserve function decreased gradually in the early stage of DM, which is related to both structural (but not reduced microvascular density) and functional abnormalities of microvessels, and is associated with increased blood glucose, reduced LV deformation, and myocardial remodeling.

Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes.

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease.

Isorhapontigenin Attenuates Cardiac Microvascular Injury in Diabetes via the Inhibition of Mitochondria-Associated Ferroptosis Through PRDX2-MFN2-ACSL4 Pathways.

Ferroptosis is a newly identified form of regulated cell death that is driven by iron overload and uncontrolled lipid peroxidation, but the role of ferroptosis in cardiac microvascular dysfunction remains unclear. Isorhapontigenin (ISO) is an analog of resveratrol and possesses strong antioxidant capacity and cardiovascular-protective effects. Moreover, ISO has been shown to alleviate iron-induced oxidative damage and lipid peroxidation in mitochondria. Therefore, the current study aimed to explore the benefits of ISO treatment on cardiac microvascular dysfunction in diabetes and the possible mechanisms involved, with a focus on ferroptosis and mitochondria. Our data revealed that ISO treatment improved microvascular density and perfusion in db/db mice by mitigating vascular structural damage, normalizing nitric oxide (NO) production via endothelial NO synthase activation, and enhancing angiogenetic ability via vascular endothelial growth factor receptor 2 phosphorylation. PRDX2 was identified as a downstream target of ISO, and endothelial-specific overexpression of PRDX2 exerted effects on the cardiac microvascular function that were similar to those of ISO treatment. In addition, PRDX2 mediated the inhibitive effects of ISO treatment on ferroptosis by suppressing oxidative stress, iron overload, and lipid peroxidation. Further study suggested that mitochondrial dynamics and dysfunction contributed to ferroptosis, and ISO treatment or PRDX2 overexpression attenuated mitochondrial dysfunction via MFN2-dependent mitochondrial dynamics. Moreover, MFN2 overexpression suppressed the mitochondrial translocation of ACSL4, ultimately inhibiting mitochondria-associated ferroptosis. In contrast, enhancing mitochondria-associated ferroptosis via ACSL4 abolished the protective effects of ISO treatment on cardiac microcirculation. Taken together, the results of the present work demonstrated the beneficial effects of ISO treatment on cardiac microvascular protection in diabetes by suppressing mitochondria-associated ferroptosis through PRDX2-MFN2-ACSL4 pathways.

La retina, una ventana de la enfermedad cerebrovascular en la diabetes

Cerebrovascular disease is one of the complications of long-term diabetes mellitus. While the structure and function of the great cerebral vessels may be more easily studied, the cerebral microcirculation is difficult to assess. However, a simple eye fundus examination with an ophthalmoscope enables to visualize the microvascular abnormalities that characterize diabetic retinopathy, which isth e most common microvascular complication of diabetes. The anatomical and functional similarity between retinal and cerebral microcirculation supports the hypothesis that alterations in retinal vascular reactivity could be considered as an early marker of cerebral microvascular dysfunction in diabetes. The initiating factor of diabetic angiopathies is endothelial dysfunction. Endothelial dysfunction results in a reduced bioavailability of nitric oxide (NO), as a consequence of decreased NO synthesis and/or increased production of free oxygen radicals that are NO scavengers. Diabetes also stimulates the production of endothelial-derived contractile factors such as superoxide anions and hydroxyl radicals, endothelin and certain cyclooxygenase (COX) derivatives. COX activation is related to a high level of oxidative stress. Oxidative stress participates in the inflammatory response involved in the diabetic vascular dysfunction. These pathogenic mechanisms have been shown in both cerebral and retinal arteries, mainly through in vitro vascular reactivity studies, suggesting that diabetes induces a profound change in microvascular regulatory mechanisms. The association between the degree of retinal perfusion, brain injuries and altered cognitive function indicates a certain parallelism in the degree of impairment of both retinal and brain circulations. In addition, prospective studies conclude that diabetic retinopathy predicts ischemic cerebrovascular disease independently of other risk factors, supporting the importance of cerebral microvascular disease in diabetics. Further research on the vascular abnormalities is needed to understand the pathogenic mechanisms underlying retinopathies and cerebrovascular disease in diabetes. In the near future, the use of fully automated methods to detect signs of retinopathy will not only facilitate the efficient evaluation of vascular changes in the retina but will also help to reduce cerebral vascular morbidity and mortality.

Micro-RNA 92a as a Therapeutic Target for Cardiac Microvascular Dysfunction in Diabetes.

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

The Diabetic Coronary Microcirculation is Regulated by MicroRNA‐21

Introduction Coronary microvascular dysfunction is particularly common among diabetes and overlaps with impaired endothelial-dependent vasodilation. These impairments are seen in early stages of the disease, but detailed mechanisms have yet to be elucidated. Nitric oxide (NO) is the major endothelial-dependent mediator of vasodilation in the healthy coronary circulation, but this mediator changes to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a risk factor for CAD, we hypothesized that a similar switch would occur. Methods Coronary arteries were isolated, and endothelial-dependent vasodilation was assessed using myography (DMT). Reactive oxygen species (ROS) in coronary endothelial cells were measured using electron paramagnetic resonance (EPR). Quantitative polymerase chain reaction (qPCR) was performed for gene expression analysis. In vivo myocardial blood flow (MBF) was measured by contrast echocardiography during increased cardiac work achieved through the administration of norepinephrine. Results Our ex vivo and in vivo data show nitric oxide synthase inhibitor (L-NAME) inhibited vasodilation in wild type (WT) mice, but the H2O2 scavenger (PEG-catalase) had no effect. In contrast, vasodilation in diabetic mice was blunted by PEG-catalase, but not L-NAME. This suggested that the mediator of coronary vasodilation switched from NO to H2O2 in diabetes. Importantly, we found that microRNA-21 (miR-21) was upregulated in diabetes and the deficiency modulated the mediator switch from NO to H2O2 in diabetic mice. Conclusions The switch in the mediator of coronary vasodilation from NO to H2O2 contributed to microvascular dysfunction in diabetes and miR-21 regulated this switch. Further genetic profiling will elucidate the pathways and mechanisms converging with miR-21 to regulate microvascular function in diabetes. This is the first mouse model that recapitulates the switch in mediator of coronary vasodilation from NO to H2O2 seen in CAD patients.

Abstract 17248: The Role of microRNA-21 in Regulating the Coronary Microcirculation in Diabetes

Introduction: Coronary microvascular dysfunction is prevalent among diabetics and intersects with deficits in endothelial-dependent vasodilation. These deficits occur early in the progression of the disease, but the mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelial-dependent mediator of vasodilation in the healthy coronary circulation, but the mediator switches to hydrogen peroxide (H 2 O 2 ) in coronary artery disease (CAD) patients. Diabetes is a risk factor for CAD, so we hypothesized that a similar switch would occur. Methods: Coronary arteries were isolated and endothelial-dependent vasodilation was assessed using myography. Quantitative polymerase chain reaction (qPCR) was performed for gene expression analysis and myocardial blood flow (MBF) was measured by contrast echocardiography. Results: Nitric oxide synthase inhibitor (L-NAME) inhibited vasodilation in wild type (WT) mice, but the H 2 O 2 scavenger (PEG-catalase) had no effect. In contrast, vasodilation in diabetic mice was blunted by PEG-catalase, but not L-NAME. This suggests that the mediator of coronary vasodilation switched from NO to H 2 O 2 in diabetes. Importantly, we found that microRNA-21 (miR-21) is upregulated in diabetes and the deficiency modulates the mediator switch from NO to H 2 O 2 in diabetic mice. Conclusions: The switch in the mediator of coronary vasodilation from NO to H2O2 contributes to microvascular dysfunction in diabetes and miR-21 regulates this switch. Further genetic profiling will elucidate the pathways and mechanisms converging with miR-21 to regulate microvascular function in diabetes. This is the first mouse model that recapitulates the switch in mediator of coronary vasodilation from NO to H 2 O 2 seen in CAD patients.

STAT3 activation in circulating myeloid-derived cells contributes to retinal microvascular dysfunction in diabetes

BackgroundLeukostasis is a key patho-physiological event responsible for capillary occlusion in diabetic retinopathy. Circulating monocytes are the main cell type entrapped in retinal vessels in diabetes. In this study, we investigated the role of the signal transducer and activator of transcription 3 (STAT3) pathway in diabetes-induced immune cell activation and its contribution to retinal microvascular degeneration.MethodsForty-one patients with type 1 diabetes (T1D) [mild non-proliferative diabetic retinopathy (mNPDR) (n = 13), active proliferative DR (aPDR) (n = 14), inactive PDR (iPDR) (n = 14)] and 13 age- and gender-matched healthy controls were recruited to the study. C57BL/6 J WT mice, SOCS3fl/fl and LysMCre/+SOCS3fl/fl mice were rendered diabetic by Streptozotocin injection. The expression of the phosphorylated human and mouse STAT3 (pSTAT3), mouse LFA-1, CD62L, CD11b and MHC-II in circulating immune cells was evaluated by flow cytometry. The expression of suppressor of cytokine signalling 3 (SOCS3) was examined by real-time RT-PCR. Mouse plasma levels of cytokines were measured by Cytometric Beads Array assay. Retinal leukostasis was examined following FITC-Concanavalin A perfusion and acellular capillary was examined following Isolectin B4 and Collagen IV staining.ResultsCompared to healthy controls, the expression of pSTAT3 in circulating leukocytes was statistically significantly higher in mNPDR but not aPDR and was negatively correlated with diabetes duration. The expression of pSTAT3 and its inhibitor SOCS3 was also significantly increased in leukocytes from diabetic mice. Diabetic mice had higher plasma levels of IL6 and CCL2 compared with control mice. LysMCre/+SOCS3fl/fl mice and SOCS3fl/fl mice developed comparative levels of diabetes, but leukocyte activation, retinal leukostasis and number of acellular capillaries were statistically significantly increased in LysMCre/+SOCS3fl/fl diabetic mice.ConclusionSTAT3 activation in circulating immune cells appears to contribute to retinal microvascular degeneration and may be involved in DR initiation in T1D.

641-P: Relationship of Sarcopenia and Microcirculation Assessed by Skin Perfusion Pressure in Patients with Type 2 Diabetes

Sarcopenia is more prevalent in diabetes than controls and is related with higher cardiovascular events such as critical limb ischemia and mortality in T2DM. Microvascular dysfunction in diabetes plays a crucial role in the development of diabetic complications. Skin perfusion pressure (SPP) using laser Doppler technique is valuable in the evaluation of microcirculation function and limb ischemia. Although previous study reported that sarcopenia is independently associated with diabetic foot disease and even it is regarded as a chronic complication of T2DM, no studies have examined the relationship of sarcopenia and microcirculation. The current analysis is an attempt to evaluate the undisclosed relationship of sarcopenia and skin perfusion pressure. We analyzed the data of 188 patients with T2DM performing laser Doppler technique for measuring SPP. Sarcopenia was defined low appendicular muscle mass adjusted for height (Men &amp;lt;7 kg/m2, women &amp;lt; 5.7kg/m2) using bioelectrical impedance analysis. We divided into two groups based on SPP: ≤50 mmHg, &amp;gt;50 mmHg. SPP below 50 mmHg was considered as impaired microcirculation. The mean age and SPP of total subjects were 51 years and 72 mmHg. A total of 26 patients (13.8%) were diagnosed with impaired function of microcirculation in limb on SPP. The prevalence of sarcopenia in total subjects was 8%. The prevalence of sarcopenia increased significantly in SPP ≤50 mmHg group than in SPP &amp;gt;50 mmHg (23% vs. 5.3%, p=0.028). Significant positive correlation was found between SPP and appendicular muscle mass adjusted for height (p=0.008). Multiple logistic regression analysis showed that patients with sarcopenia had an odds ratio of 6.1 (95% CI =1.2-31.7) for having of SPP ≤50 mmHg even after adjustment for confounding factors (p=0.032). Our results suggest that the sarcopenia is significantly associated with the presence of impaired function of microcirculation on SPP in T2DM. Disclosure C. Jung: None. D. Choi: None. C. Kim: None. J. Mok: None.

Microvascular Dysfunction and Hyperglycemia: A Vicious Cycle With Widespread Consequences.

Microvascular and metabolic physiology are tightly linked. This Perspective reviews evidence that 1) the relationship between hyperglycemia and microvascular dysfunction (MVD) is bidirectional and constitutes a vicious cycle; 2) MVD in diabetes affects many, if not all, organs, which may play a role in diabetes-associated comorbidities such as depression and cognitive impairment; and 3) MVD precedes, and contributes to, hyperglycemia in type 2 diabetes (T2D) through impairment of insulin-mediated glucose disposal and, possibly, insulin secretion. Obesity and adverse early-life exposures are important drivers of MVD. MVD can be improved through weight loss (in obesity) and through exercise. Pharmacological interventions to improve MVD are an active area of investigation.

Coronary microvascular dysfunction in diabetes: Role of HuR

Coronary microvascular dysfunction in diabetes: Role of HuR

Abstract 160: Multiscale Entropy of Photoplethysmographic Pulse Amplitudes of Bilateral Fingertips Varies by Hand Dominance and Diabetes Status

Objective: Multiscale entropy (MSE) of photoplethysmographic (PPG) pulse amplitudes may reflect cardiovascular health. In addition, poor glycemic control and weak handgrip strength are related to microvascular dysfunction in diabetes. We hypothesized that MSE of PPG pulse amplitudes between dominant and non-dominant hand may differ by diabetes status. Methods: Of a middle-to-old aged and right hand-dominant population free of prior cardiovascular disease, we matched age, sex, and weight to select the unaffected (no type 2 diabetes, n =36), the well-controlled diabetes (HbA1c &lt;8%, n =22), and the poorly-controlled diabetes (HbA1c ≥8%, n =22) groups. MSEs were calculated from simultaneous consecutive 1,500 resting PPG pulse amplitudes of bilateral index fingers. The small- and large-scale MSEs were defined as the average of scales 1 to 3 (MSE 1-3 ) and scales 4 to 10 (MSE 4-10 ), respectively. Intra- and inter-groups comparisons were performed by 1- and 2-sample t tests. Results: The inter- and intra-groups mean pulse amplitudes did not differ. In contrast, the pulse wave velocity was higher in the two diabetes groups, but was similar between two hands in each group. The dominant hand MSE 4-10 was lower in the poorly-controlled diabetes compared with the unaffected and well-controlled diabetes groups (1.25 vs. 1.52 and 1.49, p=0.001 and 0.017, respectively); whereas the non-dominant hand MSE 4-10 was lower in the well- and poorly-controlled diabetes groups than the unaffected (1.31 and 1.26 vs. 1.54, p=0.007 and 0.002 respectively). There was no intra-groups difference in MSE 4-10 , but a higher MSE 1-3 of dominant hand than that of non-dominant hand in the well-controlled diabetes (1.30 vs. 1.07, p=0.05). Conclusion: Diabetes status and hand activity are related to the MSE of PPG pulse amplitudes. The MSE indexes differed between two hands with euglycemia indicated that local physical inactivity might be an independent contributor to diabetic microvascular dysfunction.

Coronary microvascular dysfunction in diabetes mellitus.

The significance, mechanisms and consequences of coronary microvascular dysfunction associated with diabetes mellitus are topics into which we have insufficient insight at this time. It is widely recognized that endothelial dysfunction that is caused by diabetes in various vascular beds contributes to a wide range of complications and exerts unfavorable effects on microcirculatory regulation. The coronary microcirculation is precisely regulated through a number of interconnected physiological processes with the purpose of matching local blood flow to myocardial metabolic demands. Dysregulation of this network might contribute to varying degrees of pathological consequences. This review discusses the most important findings regarding coronary microvascular dysfunction in diabetes from pre-clinical and clinical perspectives.

4‐HNE modified TRPV1 dysfunction leads to loss of capsaicin‐mediated eNOS activity and microvascular dysfunction

Our lab has previously demonstrated enhanced 4‐hydroxynonenal (4‐HNE) post‐translational modification (PTM) of TRPV1 decreases TRPV1 functional expression and contributes to microvascular dysfunction in diabetes. Furthermore, recent studies have shown TRPV1 activation elicits eNOS activation and NO production. Accordingly, we hypothesize that the enhanced 4‐HNE‐mediated PTM of TRPV1 contributes to diabetic microvascular dysfunction in db/db mice via decreased eNOS activation and resultant signaling. Contrast echocardiography demonstrated that 4‐HNE decreased capsaicin mediated increases in myocardial blood flow and decreased capsaicin‐mediated relaxation in isolated coronary microvessels. TRPV1 functional analysis using calcium imaging revealed blunted capsaicin‐mediated calcium influx in the presence of 4‐HNE in isolated mouse coronary endothelial cells (MCECs). Finally, 4‐HNE treatment of MCECs decreased eNOS phosphorylation at Ser1197 leading to decreased measure in capsaicin‐mediated nitrate production. Single point mutation, of a pore region cysteine residue 621, rescued channel activity and preserved capsaicin‐mediated eNOS phosphorylation and NO production. These data suggest that TRPV1 is targeted by redox‐active substances that directly modulate channel activity at numerous sites in diabetes to decrease TRPV1 functional expression and contribute to microvascular dysfunction. The results obtained demonstrate a signaling pattern aberrant in TRPV1 regulation of vascular control in diabetes.

Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction.

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

TRPV1 residues vital to protection against 4‐HNE modification, preservation of channel activity and microvascular function

We previously demonstrated enhanced 4-hydroxynonenal (4-HNE) post-translational modification (PTM) of TRPV1 decreases TRPV1 functional expression and contributes to microvascular dysfunction in diabetes. Accordingly, we hypothesized that manipulation of residues associated with 4-HNE PTM would preserve TRPV1 function and restore vascular integrity. 4-HNE decreased capsaicin mediated increases in myocardial blood flow and capsaicin-mediated relaxation in isolated coronary microvessels. TRPV1 functional analysis using electrophysiology revealed blunted capsaicin-mediated currents in the presence of 4-HNE which were reversed by the reactive carbonyl species scavenger aminoguanidine (AGD). Using computational biology, we identified three probable residues, previously identified to be important for oxidative modification, as potential sites for 4-HNE modification. The corresponding residues, C616, C621 and C634, were mutated to alanine (individually and in combination) and subsequently we examined the effects of 4-HNE on TRPV1 currents induced by capsaicin via electrophysiology. The mutation of the three pore cysteine (individually or in combination) abrogated the effects of 4-HNE on capsaicin-mediated currents. These data suggest that TRPV1 is targeted by redox-active substances that directly modulate channel activity at numerous sites in diabetes to decrease TRPV1 functional expression and contribute to microvascular dysfunction. The results obtained demonstrate an optimal redox state is critical for a properly functioning TRPV1 channel.

IN VIVO INHIBITION OF NFAT SIGNALING RESTORES MICROVASCULAR ENDOTHELIAL FUNCTION IN DIABETIC MICE

Vascular disease is the major cause of morbidity and mortality in diabetes. We previously demonstrated that hyperglycemia activates the transcription factor nuclear factor of activated T cells (NFAT) in large and small arteries in vivo and that this leads to enhanced expression of endothelial activation and inflammatory markers. Also, that in vivo blockade of NFAT signaling abolishes the diabetes-driven aggravation of atherosclerosis in mice. Here we explored whether NFAT is involved in the development of diabetic microvascular dysfunction. We found that NFAT-dependent transcriptional activity was elevated in aorta, cerebral, retinal and skin vessels, as well as in the heart of NFAT-luciferase reporter diabetic (blood glucose >20 mmol/l) male Akita (Ins2+/-) mice when compared to littermate non-diabetic mice. Treatment of diabetic mice with the NFAT blocker A-285222 for 4 weeks (daily i.p. injections; 0.29mg/kg) resulted in improved microvascular function, as assessed by Laser Doppler Imaging and iontophoresis responses to acetylcholine (Ach) and localized heating (Ach: p<0.05; Heat: p<0.01; vs. saline treated mice; n=15-20/group). This improvement was abolished by pre-treatment with the nitric oxide synthase inhibitor L-NAME (20mg/kg i.p.). Treatment with A-285222 inhibited NFAT-transcriptional activity in the vessels but had no effect on blood glucose. We propose inhibition of NFAT activity may therefore provide a novel therapeutic modality for the treatment of diabetic microangiopathy.

Mineralocorticoid receptors: an appealing target to treat coronary microvascular dysfunction in diabetes.

Cardiovascular disease (CVD), particularly coronary vascular disease, is a primary contributor to diabetes-related morbidity and mortality worldwide. Specifically, dysfunction of coronary microcirculation is common in this patient population, often occurring in the absence of or preceding epicardial coronary atherosclerosis, thereby leading to impaired coronary blood flow (CBF) regulation and increased risk of myocardial ischemia/infarct. Clinically, coronary microvascular function can be assessed by determination of coronary flow reserve (CFR) (ratio of maximal hyperemic to basal CBF), and impaired CFR is a powerful independent correlate of cardiac mortality in diabetic patients (1). Importantly, recent evidence revealed that diabetic patients with preserved CFR (above the median) have cardiac event rates similar to nondiabetic patients (1). Thus, treatment strategies designed to restore CFR (i.e., coronary microvascular function) hold promise to reduce acute and long-term cardiac mortality in patients with diabetes. Diabetes is associated with increased activation of the renin-angiotensin-aldosterone system (RAAS), and evidence suggests that the aldosterone-binding mineralocorticoid receptor (MR) contributes to obesity and diabetes-related vascular dysfunction (2,3). Even modest elevations in circulating aldosterone levels correlate with increased acute ischemic events and cardiovascular death in diabetic patients with coronary artery disease (4). Furthermore, accumulating evidence demonstrates that inhibition of angiotensin II (AngII) action, via ACE inhibition or angiotensin receptor blockade (ARB), does not appreciably lower circulating aldosterone levels, suggesting a residual role for MR activation in CVD pathogenesis (5). This is consistent with initial clinical trials demonstrating reduced mortality with MR antagonist treatment in …