Transcutaneous Oxygen Tension as a Potential Predictor of Cardiovascular Events in Type 2 Diabetes: Comparison with ankle-brachial index

OBJECTIVE

Transcutaneous oxygen tension (TcPO2) measures tissue perfusion and is important in the management of peripheral artery disease (PAD). Ankle brachial index (ABI) is used for the diagnosis of PAD and represents a predictor of major adverse cardiovascular events (MACE), even if in diabetes its diagnostic and predictive value seems to be reduced. No study has evaluated TcPO2 as a predictor of cardiovascular events. Aim of this longitudinal study was to assess whether TcPO2 is better than ABI at predicting MACE in type 2 diabetic patients.

RESEARCH DESIGN AND METHODS

Among 361 consecutive patients with apparently uncomplicated diabetes, 67 MACE occurred during a follow-up period of 45.8 ± 23.2 months.

RESULTS

The percentage of both subjects with low ABI (≤0.9) and subjects with low TcPO2 (≤46 mmHg as measured by a receiver operating characteristic curve) was significantly (<0.001) greater among patients with than among those without MACEs (ABI 64.2 vs. 40.8; TcPO2 58.2 vs. 34%). The Kaplan-Meier method showed that both low ABI (Mantel log-rank test, 4.087; P = 0.043) and low TcPO2 (Mantel log-rank test, 33.748; P > 0.0001) were associated with a higher rate of MACEs. Cox regression analysis showed that low TcPO2 (hazard ratio 1.78 [95% CI 1.44–2.23]; P < 0.001) was a significant predictor of MACE, while ABI did not enter the model.

CONCLUSIONS

This longitudinal study showed that TcPO2 may be a potential predictor of MACE among patients with uncomplicated type 2 diabetes and that its predictive value seems to be greater than that of ABI.Diabetic patients have an increased cardiovascular risk (1,2). Indeed, cardiovascular disease is the main cause of mortality and morbidity related to diabetes, and approximately two-thirds or more of diabetic patients die of cardiovascular disease (1,2). Cardiovascular death rate of diabetic patients without prior myocardial infarction even seems to be similar to that of nondiababetic patients with prior myocardial infarction (3).Ankle brachial index (ABI), commonly used as a diagnostic test for peripheral arterial disease (PAD) (4), is considered an independent powerful marker of cardiovascular morbidity and mortality in the general population (5,6). An ABI of ≤0.90 is universally recognized as the cutoff for the diagnosis of PAD (4). However, in diabetic people the use of ABI has some important diagnostic limitations: indeed, the presence of typical medial artery calcifications causes arterial wall stiffness and a high prevalence of false-negative values (7–9). Transcutaneous oxygen tension (TcPO2) is a noninvasive method to measure tissue perfusion: it reflects very well the metabolic state of lower limbs (10,11). TcPO2 is currently used in clinical practice in the management of the vascular diabetic foot (11,12); in particular, it is important in determining amputation level, wound healing evaluation, and revascularization procedures (11,12). TcPO2 is not affected by arterial calcification and is particularly useful in evaluating PAD in diabetic patients (13); in addition, it has a good reproducibility (10–12). Nevertheless, there is not thus far a universally recognized specific cutoff of TcPO2 for the diagnosis of PAD (11). Finally, it is unknown whether TcPO2 may be a reliable marker as well of cardiovascular morbidity and mortality in diabetes. Aim of the current study was to assess whether TcPO2 is better than ABI in predicting major adverse cardiovascular events (MACEs) among diabetic patients.     

Fetuin-A levels are increased in patients with type 2 diabetes and peripheral arterial disease

OBJECTIVE

Low levels of fetuin-A, a systemic calcification inhibitor, are linked to mortality in patients on dialysis. In contrast, elevated fetuin-A is associated with cardiovascular events in non-renal patients. We investigated fetuin-A in patients with type 2 diabetes and peripheral arterial disease (PAD).

RESEARCH DESIGN AND METHODS

We studied fetuin-A in 76 patients with PAD and normal glucose metabolism (NGM-PAD) and in 129 patients with PAD and type 2 diabetes (type 2 diabetes–PAD). Additionally, 40 patients with diabetes without any complications (type 2 diabetes–non-PAD) were examined.

RESULTS

Type 2 diabetes–PAD subjects (399 ± 155 μg/ml) had significantly higher fetuin-A levels than type 2 diabetes–non-PAD subjects (247 ± 42; P < 0.001). In NGM-PAD subjects (376 ± 144), fetuin-A was significantly higher than in type 2 diabetes–non-PAD subjects (P < 0.001). Type 2 diabetes–PAD patients with mediasclerosis had lower fetuin-A than subjects without (P < 0.03). Regression analysis in type 2 diabetes–PAD subjects revealed that glycated A1C (P < 0.001) and mediasclerosis (P = 0.004) were the strongest predictors of fetuin-A. Multivariate regression revealed that a 1-SD increase in fetuin-A was associated with an odds ratio (OR) of 2.1 (95% CI 1.1–3.3; P < 0.001) for the prevalence of PAD and an OR of 1.4 (1.0–1.7, P = 0.039) for the prevalence of myocardial infarction.

CONCLUSIONS

In contrast to previous findings, fetuin-A was higher in type 2 diabetes–PAD patients than in type 2 diabetes–non-PAD patients. In NGM-PAD patients, fetuin-A was also higher than in type 2 diabetes–non-PAD patients. In type 2 diabetes–PAD patients, fetuin-A was inversely associated with mediasclerosis—the calcification process pathognomonic for diabetic PAD. This association persisted in multivariate regression, which is in line with the calcification inhibition in coronary heart or renal disease.Patients suffering from type 2 diabetes and peripheral artery disease (PAD) (type 2 diabetes–PAD) have a five times higher risk for cardiovascular mortality than patients with one disease alone (1–3). Furthermore, the risk of lower-extremity amputation is higher than in patients without diabetes (3).Fetuin-A, also known as α2-Schmid Heremans glycoprotein (ASHG), is a potent systemic calcification inhibitor (4). Fetuin-A knockout mice develop severe calcification of various organs (4). In a cross-sectional study, low levels of fetuin-A were associated with cardiovascular mortality in patients on dialysis (5). In addition, low fetuin-A has been linked to vascular calcification (6) and flow-limiting aortic stenosis (7).Fetuin-A interacts with the insulin receptor tyrosine kinase and induces insulin resistance in rodents (8,9). Stefan et al. (10) demonstrated in a prospective case-cohort study that elevated fetuin-A is an independent risk factor for developing diabetes. Contrariwise to renal (dialysis) patients, several studies showed that high levels of fetuin-A were associated with atherosclerosis and its manifestations in non-renal patients (11–13). Likewise, high levels of fetuin-A were linked to myocardial infarction and ischemic stroke (12). This possible involvement of fetuin-A in the pathogenesis of cardiovascular disease has been confirmed by a recent trans-European cohort study with 2,520 patients (13). Thus, it seems that high levels of fetuin-A are associated with atherosclerosis and its manifestations in non-renal patients.In contrast to the latter findings, a recent article (14) suggested that fetuin-A levels in a non-dialysis condition are lower in type 2 diabetes–PAD patients (n = 38) than in patients with diabetes alone.However, the role of fetuin-A and its involvement in atherosclerosis seems to be very complex and yet not understood. The situation is even more complex in patients with type 2 diabetes–PAD, who generally suffer from advanced/systemic atherosclerosis (1–3,15). In those high-risk patients, up to 30% show mediasclerosis (2,15). The aim of this study was to investigate fetuin-A levels in patients with type 2 diabetes with or without PAD in comparison with PAD patients with diabetes.     

Long-Term Cardiovascular Risk in Type 2 Diabetic Compared With Nondiabetic First Acute Myocardial Infarction Patients: A population-based cohort study in southern Europe

OBJECTIVE

The aim of this study was to determine whether long-term cardiovascular risk differs in type 2 diabetic patients compared with first acute myocardial infarction patients in a Mediterranean region, considering therapy, diabetes duration, and glycemic control.

RESEARCH DESIGN AND METHODS

A prospective population-based cohort study with 10-year follow-up was performed in 4,410 patients aged 30–74 years: 2,260 with type 2 diabetes without coronary heart disease recruited in 53 primary health care centers and 2,150 with first acute myocardial infarction without diabetes recruited in 10 hospitals. We compared coronary heart disease incidence and cardiovascular mortality rates in myocardial infarction patients and diabetic patients, including subgroups by diabetes treatment, duration, and A1C.

RESULTS

The adjusted hazard ratios (HRs) for 10-year coronary heart disease incidence and for cardiovascular mortality were significantly lower in men and women with diabetes than in myocardial infarction patients: HR 0.54 (95% CI 0.45–0.66) and 0.28 (0.21–0.37) and 0.26 (0.19–0.36) and 0.16 (0.10–0.26), respectively. All diabetic patient subgroups had significantly fewer events than myocardial infarction patients: the HR of cardiovascular mortality ranged from 0.15 (0.09–0.26) to 0.36 (0.24–0.54) and that of coronary heart disease incidence ranged from 0.34 (0.26–0.46) to 0.56 (0.43–0.72).

CONCLUSIONS

Lower long-term cardiovascular risk was found in type 2 diabetic and all subgroups analyzed compared with myocardial infarction patients. These results do not support equivalence in coronary disease risk for diabetic and myocardial infarction patients.The prevalence of diabetes is reaching epidemic proportions in developed countries (1). For example, the U.S. has 18 million diabetic patients, Spain has >2 million diabetic patients, and management of the disease costs >$132 and >$3.3 billion per year, respectively (2).Some studies (3–5), several of them with great influence on important guidelines for cardiovascular prevention (3), suggest that the cardiovascular risk of diabetic patients is similar to that of coronary heart disease secondary prevention patients. Other reports, however, do not confirm these observations (6–10).Part of the discrepancy may stem from differences in the duration of diabetes, type of treatment, and baseline glucose control of diabetic patients included in the studies (3–5). These limit comparability, given the fact that time of evolution and treatment required to attain appropriate glycemic control are key determinants of prognosis (10–16).Among population-based cohort studies that compared the prognosis of diabetic patients with that of myocardial infarction patients without diabetes (3–10), only two analyzed the role of diabetes duration (11,12). Even these studies did not include unstable angina among the end points and risk was not stratified by type of treatment. To our knowledge, the effect of type 2 diabetes on coronary heart disease incidence has barely been studied in southern Europe, a region known for low cardiovascular mortality (17). The aim of this study was to determine whether long-term cardiovascular risk differed between type 2 diabetic patients and first acute myocardial infarction patients and to assess the influence of diabetes duration, type of treatment, and glycemic control at baseline.     

No loss of chance of diabetic retinopathy screening by endocrinologists with a digital fundus camera

OBJECTIVE

To compare the efficacy of the diabetic retinopathy (DR) screening with digital camera by endocrinologists with that by specialist and resident ophthalmologists in terms of sensitivity, specificity, and level of “loss of chance.”

RESEARCH DESIGN AND METHODS

In a cross-sectional study, 500 adult diabetic patients (1,000 eyes) underwent three-field retinal photography with a digital fundus camera following pupillary dilatation. Five endocrinologists and two ophthalmology residents underwent 40 h of training on screening and grading of DR and detection of associated retinal findings. A κ test compared the accuracy of endocrinologist and ophthalmology resident screening with that performed by experienced ophthalmologists. Screening efficiency of endocrinologists was evaluated in terms of “loss of chance,” i.e., missed diagnoses that required ophthalmologist referrals.

RESULTS

The mean weighted κ of DR screening performed by endocronologists was similar to that of ophthalmology residents (0.65 vs. 0.73). Out of 456 DR eyes, both endocrinologists and ophthalmology residents misdiagnosed only stage 1 DR (36 and 14, respectively), which did not require ophthalmologist referral. There were no significant differences between endocrinologists and ophthalmology residents in terms of diabetic maculopathy and incidental findings except for papillary cupping and choroidal lesions, which were not the main purpose of the study or of the training.

CONCLUSIONS

The endocrinologist with specific training for DR detection using a three-field digital fundus camera with pupillary dilatation can perform a reliable DR screening without any loss of chance for the patients when compared with identical evaluation performed by experienced ophthalmologists.Diabetic retinopathy (DR) is one of the main causes of blindness in industrialized nations (1). The worldwide prevalence of diabetes in adults is estimated to rise to 7.7%, affecting 439 million adults by 2030 (2). In France, the increasing number of patients with diabetes, coupled with the lack of a national screening program, results in a steady rise in the visual handicaps related to the disease (3).Annual screening of DR is recommended as an effective approach to prevent visual loss related to diabetes (4,5). Currently, digital nonmydriatic fundus photography is increasingly used as a method of screening for ophthalmologists worldwide (5–7). According to consensus classifications (4,5), DR at a stage higher than 1 needs further ophthalmological management. Despite these recommendations, only 30% of the diabetic patients in France undergo DR screening each year. Partly, this is due to the lack of ophthalmologists and insufficient awareness about the visual consequences of the disease (3,8). The situation is slowly changing after implementation of telemedical screening networks using digital fundus photography (9–11). Further increase in screening coverage can be achieved with the involvement of allied medical professionals.Since the 1980s, the concept of “loss of chance” has emerged in medicine and law. The misdiagnosis during DR screening can lead to a loss of chance for patients requiring referral to an ophthalmologist for further examinations and management (12,13).Two studies have shown that screening performed by an endocrinologist using an ophthalmoscope (14) and a mydriatic camera (15), respectively, were reliable, although they didn’t evaluate the loss of chance. Furthermore, no endocrinologists’ team approach was evaluated so far.This clinical research trial has been designed to compare the efficacy of the DR screening with digital camera by a team of previously trained endocrinologists (7) with that of residents and specialist ophthalmologists, in terms of sensitivity, specificity and level of “loss of chance.”     

Rates of Progression in Diabetic Retinopathy During Different Time Periods: A systematic review and meta-analysis

OBJECTIVE

This meta-analysis reviews rates of progression of diabetic retinopathy to proliferative diabetic retinopathy (PDR) and/or severe visual loss (SVL) and temporal trends.

RESEARCH DESIGN AND METHODS

This systematic literature review and meta-analysis of prospective studies assesses progression of retinopathy among diabetic patients without treatment for retinopathy at baseline. Studies published between 1975 to February 2008 were identified. Outcomes of interest were rates of progression to PDR and/or SVL. Pooled baseline characteristics and outcome measures were summarized using weighted averages of counts and means. Baseline characteristics and outcomes were compared between two periods: 1975–1985 and 1986–2008.

RESULTS

A total of 28 studies comprising 27,120 diabetic patients (mean age 49.8 years) were included. After 4 years, pooled incidence rates for PDR and SVL were 11.0 and 7.2%, respectively. Rates were lower among participants in 1986–2008 than in 1975–1985. After 10 years, similar patterns were observed. Participants in 1986–2008 studies had lower proportions of PDR and non-PDR at all time points than participants in 1975–1985 studies.

CONCLUSIONS

Since 1985, diabetic patients have lower rates of progression to PDR and SVL. These findings may reflect an increased awareness of retinopathy risk factors; earlier identification and initiation of care for patients with retinopathy; and improved medical management of glucose, blood pressure, and serum lipids. Differences in baseline characteristics, particularly in the prevalence and severity of retinopathy, could also have contributed to these temporal differences.Diabetes affects more than 170 million individuals worldwide (1,2), and diabetic retinopathy is the most frequent cause of visual impairment among working-age individuals (3,4). In the last 3 decades, a relative decline in rates of diabetic retinopathy has been suggested by some studies, (5–8) possibly reflecting improved patient and physician awareness, screening, and prevention, as well as better management of diabetes (9). In 1985, the Early Treatment Diabetic Retinopathy Study (ETDRS) demonstrated the effectiveness of laser photocoagulation (10,11). Systemic control of both hyperglycemia and hypertension was shown to be important in the Diabetes Control and Complications Trial (DCCT) and the UK Prospective Diabetes Study (UKPDS) in the 1990s (12,13). Findings from these trials, other studies, and clinical practice guidelines may have led to increased public awareness to diabetes risk factors and a shorter time from onset to diagnosis, potentially altering the rates of diabetic retinopathy progression (9,14).Understanding the natural history of diabetic retinopathy is also important for estimating sample size for testing new interventions in clinical trials. Already, inadequate sample size estimations may have resulted in underpowered trials (15). Traditionally, progression rates from the ETDRS and the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) were used for sample size calculations (16–22). However, these studies were conducted almost 30 years ago. Contemporary estimates for diabetic retinopathy progression are clearly needed, some of which may, in part, be provided by more recent studies, such as the Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy (DIRECT) trial (23,24).In this systematic review and meta-analysis, we summarized the best available evidence to provide contemporary data on the clinical course of diabetic retinopathy and to examine potential differences in rates of diabetic retinopathy progression over time.     

Inflammatory, Hemostatic, and Other Novel Biomarkers for Diabetic Retinopathy: The Multi-Ethnic Study of Atherosclerosis

OBJECTIVE

There are conflicting data regarding relationships of systemic biomarkers of inflammation, hemostasis, and homocysteine with diabetic retinopathy. We examined these relationships in the Multi-Ethnic Study of Atherosclerosis.

RESEARCH DESIGN AND METHODS

A total of 921 participants with diabetes were included. Diabetic retinopathy was graded from retinal photographs. We defined two outcomes: any diabetic retinopathy and vision-threatening diabetic retinopathy (severe nonproliferative diabetic retinopathy or worse). Systemic markers analyzed were C-reactive protein, homocysteine, fibrinogen, plasmin-α₂-antiplasmin complex (PAP), interleukin-6, d-dimer, factor VIII, serum creatinine, and urinary albumin-to-creatinine (UAC) ratio.

RESULTS

Prevalence of diabetic retinopathy was 33.2% and vision-threatening diabetic retinopathy 7.1%. After adjusting for established risk factors (diabetes duration, A1C, systolic blood pressure, waist-to-hip ratio, and use of diabetes medications), fibrinogen (odds ratio 1.14 [95% CI 1.01–1.32], P = 0.05) and PAP (1.25 [1.05–1.50], P = 0.01) were associated with any diabetic retinopathy, while PAP (1.54 [1.13–2.11], P = 0.007) and homocysteine (1.57 [1.16–2.11], P = 0.003) were associated with vision-threatening diabetic retinopathy. Only PAP remained significant after additional adjustment for serum creatinine and UAC ratio. Area under receiver-operator characteristic curve (AUROC) for diabetic retinopathy was constructed for established and novel risk factors. Established risk factors accounted for a 39.2% increase of the AUROC, whereas novel markers (fibrinogen, PAP, homocysteine, serum creatinine, and UAC ratio) only accounted for an additional 2.2%.

CONCLUSIONS

There were few associations of novel markers of inflammation, hemostasis, and homocysteine with diabetic retinopathy after controlling for established risk factors. These data suggest that there is limited clinical use of these biomarkers for prediction of diabetic retinopathy.Diabetic retinopathy is the leading cause of blindness in working-age individuals (1). There is increasing evidence that established risk factors for diabetic retinopathy (2,3), including duration of diabetes, hyperglycemia, and hypertension, only explain a limited amount of the variance in the risk of diabetic retinopathy (1). Furthermore, the underlying pathogenesis of diabetic retinopathy remains inadequately understood (4). This has resulted in examination of the relation of novel risk markers such as inflammation (e.g., C-reactive protein [CRP]), markers of hemostatic disturbances (e.g., fibrinogen levels), and hyperhomocysteinemia to diabetic retinopathy. However, to date, the relations of these factors to diabetic retinopathy have not been consistent (5–17). The reasons for these inconsistencies may be due, in part, to differences in study sample and definitions of diabetic retinopathy (e.g., clinical versus photograph grading) and failure in some studies to make adequate adjustments for traditional risk factors such as glycemic control and hypertension. Thus, it remains unclear if there is a role for the use of these systemic markers as additional clinical tests to identify individuals at high risk of diabetic retinopathy. In this study, we evaluated the relationship of a range of inflammatory, hemostatic, and novel vascular markers with diabetic retinopathy, while controlling for traditional risk factors, in a large multiethnic population.     

Can the Retinal Screening Interval Be Safely Increased to 2 Years for Type 2 Diabetic Patients Without Retinopathy?

OBJECTIVE

In the U.K., people with diabetes are typically screened for retinopathy annually. However, diabetic retinopathy sometimes has a slow progression rate. We developed a simulation model to predict the likely impact of screening patients with type 2 diabetes, who have not been diagnosed with diabetic retinopathy, every 2 years rather than annually. We aimed to assess whether or not such a policy would increase the proportion of patients who developed retinopathy-mediated vision loss compared with the current policy, along with the potential cost savings that could be achieved.

RESEARCH DESIGN AND METHODS

We developed a model that simulates the progression of retinopathy in type 2 diabetic patients, and the screening of these patients, to predict rates of retinopathy-mediated vision loss. We populated the model with data obtained from a National Health Service Foundation Trust. We generated comparative 15-year forecasts to assess the differences between the current and proposed screening policies.

RESULTS

The simulation model predicts that implementing a 2-year screening interval for type 2 diabetic patients without evidence of diabetic retinopathy does not increase their risk of vision loss. Furthermore, we predict that this policy could reduce screening costs by ∼25%.

CONCLUSIONS

Screening people with type 2 diabetes, who have not yet developed retinopathy, every 2 years, rather than annually, is a safe and cost-effective strategy. Our findings support those of other studies, and we therefore recommend a review of the current National Institute for Health and Clinical Excellence (NICE) guidelines for diabetic retinopathy screening implemented in the U.K.Diabetic retinopathy is a serious complication for people with diabetes that can lead to blindness or severe vision loss (1). Although retinopathy cannot be cured, its progression can be slowed or halted, and laser treatment can prevent visual loss if offered promptly at the proliferative stage (2). Diabetic digital retinal photography is an effective method of detecting the onset of treatable retinopathy (3). National Institute for Health and Clinical Excellence (NICE) guidelines in the U.K. currently recommend that people with diabetes are screened for retinopathy annually, or every 3–6 months for those patients who have developed beyond mild background retinopathy or who are at higher risk of progression (4). This also forms part of the National Screening Committee’s policy of a national screening program for diabetes in the U.K. (5).The development of retinopathy may take decades (6), and the annual screening of all diabetic patients may therefore incur considerable cumulative health service cost and patient inconvenience that, for some, may be unjustified (7). With a significant increase in diabetes incidence forecast in the U.K. (8), it is imperative that screening policies for diabetes complications are cost-effective and practicable. It is therefore prudent to ask whether it would be cost-effective and safe to screen diabetic patients for retinopathy less frequently (7), particularly in light of more recent evidence that suggests an overall decline in the rates at which people with diabetes are developing vision-threatening retinopathy (9,10).We undertook a collaborative project with the Royal Devon and Exeter National Health Service (NHS) Foundation Trust (henceforth referred to simply as Royal Devon and Exeter), which carries out annual retinopathy screening for a population of ∼20,000 patients across Devon. Using simulation modeling, we assessed the potential impact of implementing a 2-year retinopathy screening interval for those patients without retinopathy and who have type 2 diabetes. Diabetic retinopathy may progress more quickly in patients with type 1 diabetes (1,11–13), and although it has been shown that less frequent screening may be feasible for type 1 patients (14), we focus on the lower-risk type 2 diabetic patient group in this study. Previous studies have looked at the cost-effectiveness of longer screening intervals for diabetic retinopathy screening across all patients in a population (7,15,16), but we assessed an increased screening interval solely for those patients who have not yet been diagnosed with diabetic retinopathy. These patients represent ∼40% of the type 1 and type 2 diabetic population screened by Royal Devon and Exeter, and reductions in the frequency with which they are screened could therefore lead to large potential cost and resource savings.     

A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.NK cells represent a distinct lineage of lymphocytes that are able to kill a variety of tumor (1), virus-infected (2), bone marrow transplanted (3), and allogeneic target cells (4). NK cells do not express T cell receptors or immunoglobulins and are apparently normal in mice with defects in the recombinase machinery (5, 6).Our knowledge about NK cell specificity has increased considerably in the last years. NK cells can probably interact with target cells by a variety of different cell surface molecules, some involved in cell adhesion, some activating the NK cytolytic program (7, 8), and other ones able to inhibit this activation by negative signaling (as reviewed in reference 9).A common feature of several inhibitory NK receptors is the capability to bind MHC class I molecules (10, 11), as predicted by the effector inhibition model within the missing self hypothesis of recognition by NK cells (12–14). Interestingly, the MHC class I receptors identified so far belong to different gene families in mouse and man; these are the p58/p70/NKAT or killer cell inhibitory receptors (KIR)¹ of the immunoglobulin superfamily in man and the Ly49 receptors of the C-type lectin family in the mouse. There is also evidence that MHC class I molecules can be recognized as triggering signals in NK cells of humans, rats as well as mice (13). The inhibitory receptors allow NK cells to kill tumor or normal cell targets with deficient MHC class I expression (12, 14). This does not exclude that other activating pathways can override inhibition by MHC class I molecules (15) and, even in their absence, there must be some activating target molecules that initiate the cytolytic program. Several surface molecules are able to mediate positive signals in NK cells. Some of these structures, like NKRP1 (16), CD69 (17), and NKG2 (18) map to the NK complex region (NKC) of chromosome 6 in mice and of chromosome 12 in humans (13). CD2 (19) and CD16 (20) molecules can also play a role in the activation pathway.NK cells resemble T cells in many respects, both may arise from an immediate common progenitor (21, 22), and share the expression of several surface molecules (23). NK cells produce cytokines resembling those secreted by some helper T cell subsets (24) and contain CD3 components in the cytoplasm (21). The expression of some surface structures, involved in TCR-dependent T cell costimulation, like CD28 in human (25), has been described on NK cells, but the functional relevance of these molecules for NK activation processes has not been fully established.Another T cell molecule of interest is CD40L, which interacts with CD40, a 50-kD membrane glycoprotein expressed on B cells (26), dendritic cells (27), and monocytes (28). CD40 is a member of the tumor necrosis factor/nerve growth factor receptor family (29) which includes CD27 (30), CD30 (31), and FAS antigen (32). Murine and human forms of CD40L had been cloned and found to be membrane glycoproteins with a molecular mass of ∼39 kD induced on T cells after activation (33). Also mast cells (34), eosinophils (35), and B cells (36) can be induced to express a functional CD40L. The CD40L–CD40 interaction has been demonstrated to be necessary for T cell–dependent B cell activation (33, 37). Mutations in the CD40L molecule cause a hyper-IgM immunodeficiency condition in man (38, 39, 40). On the other hand, CD40–CD40L interactions also orchestrate the response of regulatory T cells during both their development (41, 42) and their encounter with antigen (43, 44).NK cells have also been suggested to play a role in B cell differentiation and immunoglobulin production (45). Therefore, it was of interest to investigate whether NK cells could use a CD40-dependent pathway in their interactions with other cells. Therefore, we have investigated the ability of target cells expressing CD40 to induce activation of NK cytotoxicity.     

TRAF-interacting Protein (TRIP): A Novel Component of the Tumor Necrosis Factor Receptor (TNFR)- and CD30-TRAF Signaling Complexes That Inhibits TRAF2-mediated NF-κB Activation

Through their interaction with the TNF receptor–associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor–TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-κB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor–proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.Members of the TNF receptor (TNFR)¹ superfamily play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1). Whether the signals induced by a given receptor leads to cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1). The cytoplasmic domains of these receptors lack intrinsic catalytic activity and also exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNFR1 that share a significant homology within an 80–amino acid region of their cytoplasmic tails (called the “death domain”; 2, 3). Therefore, it is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex (1).Indeed, several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4–19). Recent biochemical and molecular studies showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD (MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).The TNFR family members can also recruit a second class of signal transducers called TRAFs (TNFR-associated factor), some of which are responsible for the activation of NF-κB or JNK (9, 20, 22). TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30, or LT-βR (4, 5, 10, 11, 15, 23–27). These receptors interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15, 24–27). To date, five members of the TRAF family have been identified as signaling components of the TNFR family members. All TRAF members contain a conserved TRAF domain, ∼230 amino acids in length, that is used for either homo- or heterooligomerization among the TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23–25, 28). In addition to the TRAF domain, most of the TRAF family members contain an NH₂-terminal RING finger and several zinc finger structures, which appear to be important for their effector functions (4, 5, 10, 11, 23–25).Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF-κB activation induced by two TNF receptors, CD40 and CD30 (9, 28–30). TRAF5 was also implicated in NF-κB activation mediated by LTβR (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in the TNFR family–mediated signal transduction is not clear. They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the cellular inhibitor of apoptosis protein (c-IAP) family to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs may regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of IL-1 receptor (IL1R)–signaling complex, in which it mediates the activation of NF-κB by IL-1R (31). Since TRAFs form homo- or heterooligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, it needs to be determined whether TRAF proteins will also mediate the apoptotic signals induced by the “death-domain-less” members of the TNFR superfamily (1, 27, 32–36).Here we report the isolation and characterization of a novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein). TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF-κB activation. Biochemical studies indicate that TRIP associates with the TNFR2 or CD30 receptor complex via its interaction with TRAF proteins, suggesting a model which can explain why the ligation of these receptors can promote different cell fates: proliferation or death.     

Diabetes and risk of prostate cancer: a study using the National Health Insurance

OBJECTIVE

The link between diabetes and prostate cancer is rarely studied in Asians.

RESEARCH DESIGN AND METHODS

The trend of age-standardized prostate cancer incidence in 1995–2006 in the Taiwanese general population was calculated. A random sample of 1,000,000 subjects covered by the National Health Insurance in 2005 was recruited. A total of 494,630 men for all ages and 204,741 men ≥40 years old and without prostate cancer at the beginning of 2003 were followed to the end of 2005. Cumulative incidence and risk ratio between diabetic and nondiabetic men were calculated. Logistic regression estimated the adjusted odds ratios for risk factors.

RESULTS

The trend of prostate cancer incidence increased significantly (P < 0.0001). The cumulative incidence markedly increased with age in either the diabetic or nondiabetic men. The respective risk ratio (95% CI) for all ages and age 40–64, 65–74, and ≥75 years was 5.83 (5.10–6.66), 2.09 (1.60–2.74), 1.35 (1.07–1.71), and 1.39 (1.12–1.71). In logistic regression for all ages or for age ≥40 years, age, diabetes, nephropathy, ischemic heart disease, dyslipidemia, living region, and occupation were significantly associated with increased risk, but medications including insulin and oral antidiabetic agents were not.

CONCLUSIONS

Prostate cancer incidence is increasing in Taiwan. A positive link between diabetes and prostate cancer is observed, which is more remarkable in the youngest age of 40–64 years. The association between prostate cancer and comorbidities commonly seen in diabetic patients suggests a more complicated scenario in the link between prostate cancer and diabetes at different disease stages.The association between diabetes and prostate cancer has been inconsistently reported, even though two meta-analyses suggested that diabetic patients have a lower risk of prostate cancer of 9% (1) and 16% (2), respectively.While the two meta-analyses were examined, many studies were case-control and only three focused on the follow-up of cohorts of diabetic patients (3–5). Among the three cohorts, the cases of prostate cancer were 9 (3), 498 (4), and 2,455 (5), respectively; and only the last (5) showed a significant 9% risk reduction in diabetic patients. Except for the first study being conducted in residents with diabetes in Rochester, Minnesota (3), the diabetic patients in the other two were from hospitalized patients in Denmark (4) and Sweden (5), respectively. The meta-analyses have limitations including a mixture of case-control and cohort designs, a mixture of incident and dead cases, a small number of prostate cancer in most studies, and different sources of subjects with potential selection bias. Although the contamination of type 1 diabetes is possibly minimal because >90% of overall patients have type 2 diabetes, residual confounding could not be excluded if the two types of diabetes are not differentiated.Although some recent studies still suggested a lower risk of prostate cancer in diabetic patients including Caucasians (6,7), Iranians (8), Israelis (9), African Americans, Native Hawaiians, and Japanese Americans (6), the lower risk in African Americans and Native Hawaiians (6) was not significant. Two Japanese studies did not find any significant association (10,11). The Ohsaki Cohort Study suggested that diabetes was not predictive for total prostate cancer, but diabetic patients did show a higher risk of advanced cancer (11).Because diabetic patients are prone to develop cancer involving pancreas, liver, breast, colorectum, bladder, and endometrium (12–15) and the protective effect of diabetes on prostate cancer requires confirmation, this study evaluated the possible link between diabetes and prostate cancer, and the potential risk factors, by using the reimbursement database of the National Health Insurance (NHI) in Taiwan.     

Effect of diabetes on severity and hospital mortality in patients with acute pancreatitis: a national population-based study

OBJECTIVE

Diabetes may increase the risk of acute pancreatitis (AP). We aimed to further investigate whether diabetes may also adversely affect outcomes of patients with AP.

RESEARCH DESIGN AND METHODS

In this retrospective cohort study, we compared 18,990 first-attack AP with diabetes to 37,980 matched control subjects from Taiwan’s National Health Insurance Research Database between 2000 and 2009. Primary outcomes were development of severe AP, defined by a modified Atlanta classification scheme, and hospital mortality. Analyses were performed using univariable and multivariable logistic regression model with generalized estimating equations accounting for hospital clustering effect.

RESULTS

After baseline characteristics were adjusted, AP patients with diabetes had a higher risk of a severe attack than their nondiabetic counterparts (adjusted odds ratio [OR] 1.21, 95% CI 1.16–1.26). When severity criteria were analyzed individually, diabetic AP patients had a 58% higher risk of intensive care unit admission and a 30% higher risk of local complications, but a 16% lower risk of gastrointestinal bleeding, than AP patients without diabetes. The risk of organ failure at least one system) was similar between the two groups. Conversely, AP patients with diabetes were associated with a lower risk of hospital mortality (adjusted OR 0.77, 95% CI 0.65–0.91).

CONCLUSIONS

Although diabetes may adversely affect the disease process of AP, it seems to protect patients from AP-related mortality.Acute pancreatitis (AP) is an acute inflammatory disease of the pancreas. The local inflammation is usually self-limited within a few days, but it can be destructive and cause a severe local complication and/or systemic reaction leading to organ failures and death. Although the case-fatality rate has been decreasing over the decades (1,2), severe cases still carry a high mortality (20–50%) and consume nearly half of the resources and costs incurred by all patients with AP (3). Accordingly, many efforts have been made to identify correlates of severity and predictors for mortality in patients with AP (4–6).In addition to older people (7), patients with certain comorbidities, such as obesity (8), hypertriglyceridemia (9), chronic renal failure (10), and systemic lupus erythematosus (11), are shown to be associated with greater risk of not only the incidence but also the severity and mortality of AP. Among various comorbidities, diabetes mellitus is relatively common in patients with AP; the prevalence was 11% in Japan (12), 17.7% in California (U.S.), (13) and 19.3% in Taiwan (3). These figures are expected to continuously increase in the future because diabetic patients not only are at risk for developing AP (14–16) but also are growing in prevalence worldwide (17). Nonetheless, the effect of diabetes on outcomes of patients with AP has not been adequately studied, and the results of available reports are inconsistent (13,18). For example, Frey and colleagues examined the effect of comorbidities on patients with AP and found that diabetes was not associated with early mortality (13), whereas Graham and coworkers assessed the effect of diabetes on critically ill patients and showed a reduced risk of hospital mortality in a subgroup patients with AP (18). In both studies, however, the effect of diabetes was not specifically examined and detailed analyses were not performed (13,18).In a recent national population-based study on Taiwanese patients with first-attack AP, we found that the prevalence of diabetes increased from 15.6% in 2000 to 2001 to 19.7% in 2008 to 2009 (1). In this study, we used the same cohort (1) to further investigate the effect of diabetes on outcomes of these patients. Because diabetic patients are likely to have a higher comorbid burden and hence a poorer reserve for acute illnesses, we hypothesized that diabetes is associated with a higher risk of severe attacks and hospital mortality in adult patients with first-attack AP.     

VEGF Levels in Plasma in Relation to Platelet Activation,Glycemic Control,and Microvascular Complications in Type 1 Diabetes

OBJECTIVE

Increased levels of vascular endothelial growth factor (VEGF) in human plasma samples have suggested that circulating VEGF is a cause of endothelial dysfunction in diabetes mellitus. However, artificial release of VEGF from platelets as a source of VEGF in plasma samples, as also occurs in serum samples, has not been ruled out in these studies.

RESEARCH DESIGN AND METHODS

We determined VEGF levels in plasma collected in both citrate and PECT, a medium that inactivates platelets, in a cross-sectional cohort of 21 healthy subjects and 64 patients with type 1 diabetes. In addition, we evaluated whether VEGF levels in both types of plasma correlated with the presence of diabetes, glycemic control, markers of in vivo or ex vivo platelet activation, and degree of diabetic retinopathy and nephropathy.

RESULTS

VEGF levels were invariably low in PECT plasma of both nondiabetic and diabetic subjects and were unrelated to any other diabetes-related variable studied. In contrast, VEGF levels in citrate plasma were 150% higher in diabetic patients than in control subjects and correlated with diabetes-related variables. Multiple linear regression analysis showed that levels of platelet factor 4, a marker for ex vivo platelet activation, and HbA_1c were the independent predictors of VEGF levels in citrate plasma. Platelet activation, in vivo and ex vivo, was similar in diabetic persons and control subjects.

CONCLUSIONS

Like serum, citrate plasma is not suitable for reliable measurements of circulating VEGF. The low levels of VEGF in vivo, as represented by measurements in PECT plasma in our study, do not support a role of circulating VEGF in endothelial dysfunction in type 1 diabetes. Higher levels of VEGF in citrate plasma samples of diabetic persons do not represent the in vivo situation, but mainly originate from higher artificial ex vivo release from platelets correlating with the degree of glycemic control.Type 1 diabetes mellitus (DM) induces systemic endothelial dysfunction, even before clinical microvascular or macrovascular angiopathies become manifest (1–5). It is characterized by increased vascular permeability and elevated plasma levels of endothelium-derived proteins like von Willebrand factor (vWF) (2,3). Endothelial dysfunction is related to increased glycemia, the most important risk factor for clinical diabetic angiopathies (3–5), but its pathogenesis is unclear.Vascular endothelial growth factor (VEGF)-A is a potent angiogenic cytokine, released by hypoxic cells, activated platelets, leukocytes, and cancer cells (6–13). It increases vascular permeability in vivo and activates endothelial cells in vitro, leading to protein kinase C activation and release of several proteins for which plasma levels are elevated in vivo due to diabetic endothelial dysfunction (6,14). In cultured endothelial cells, VEGF production is induced by high levels of glucose and advanced glycation end products (15–18). Taken together, VEGF may play an important role in the pathogenesis of endothelial dysfunction (14,16,18). In support of this notion, VEGF plasma levels were found to be higher in diabetic persons than in control subjects (19–22), and a correlation of plasma VEGF levels with both diabetic nephropathy (DN) and proliferative retinopathy has been reported (23–25).As VEGF is released by activated platelets (7–10), as occurs in serum samples, platelet activation during blood collection could be an artificial source of VEGF in plasma samples as well, but this possibility was not explored in the studies mentioned (19,23,24). VEGF levels in plasma samples may correlate with blood platelet activation at the time of blood sampling rather than represent the steady-state plasma levels of VEGF in vivo. This may explain conflicting results in studies of VEGF plasma levels in DM and other conditions with endothelial dysfunction (14,20–22,26–29).Artificial ex vivo platelet activation during blood collection procedures can be largely inhibited by using anticoagulants to which a mixture of prostaglandin E₁ and theophylline has been added (e.g., PECT medium) (30). When combined with measurements for platelet factor 4 (PF4), a marker for ex vivo artificial platelet activation in blood samples (31), and β-thromboglobulin (β-TG), a marker for in vivo platelet activation (31), PECT plasma allows a more accurate estimation of the actual levels of VEGF circulating in vivo. Using this approach, we have shown in a recent study that VEGF levels in the circulation of patients with metastasized cancer are very low, in contrast to many previous reports that were based on VEGF levels in citrate or EDTA plasma analyses (32).In the current study, we compared VEGF measurements in blood samples of diabetic persons and control subjects collected in citrate and PECT medium. We investigated: 1) whether VEGF levels found in citrate plasma levels were different from those found in PECT plasma; and 2) whether VEGF levels in citrate and PECT plasma correlate with parameters of diabetes and endothelial dysfunction such as HbA_1c levels, glucose levels, vWF levels, or with the presence of diabetic retinopathy (DR) and DN.     

Corneal Nerve Loss Detected With Corneal Confocal Microscopy Is Symmetrical and Related to the Severity of Diabetic Polyneuropathy

OBJECTIVE

To establish if corneal nerve loss, detected using in vivo corneal confocal microscopy (IVCCM), is symmetrical between right and left eyes and relates to the severity of diabetic neuropathy.

RESEARCH DESIGN AND METHODS

Patients (n = 111) with type 1 and type 2 diabetes and 47 age-matched healthy control subjects underwent detailed assessment and stratification into no (n = 50), mild (n = 26), moderate (n = 17), and severe (n = 18) neuropathy. IVCCM was performed in both eyes and corneal nerve fiber density (CNFD), branch density (CNBD), and fiber length (CNFL) and the tortuosity coefficient were quantified.

RESULTS

All corneal nerve parameters differed significantly between diabetic patients and control subjects and progressively worsened with increasing severity of neuropathy. The reduction in CNFD, CNBD, and CNFL was symmetrical in all groups except in patients with severe neuropathy.

CONCLUSIONS

IVCCM noninvasively detects corneal nerve loss, which relates to the severity of neuropathy, and is symmetrical, except in those with severe diabetic neuropathy.Diabetic sensorimotor polyneuropathy (DSPN) is a length-dependent, symmetrical neuropathy with initial involvement of sensory and autonomic nerve fibers (NFs), followed by motor nerve involvement (1). It is the most common long-term complication of diabetes and is the main initiating factor for foot ulceration and lower extremity amputation with substantial associated healthcare costs (2). Conventional techniques of electrophysiology and quantitative sensory testing along with an assessment of neurological disability offer a relatively robust means of defining neuropathic severity (3) but have limitations in detecting the earliest stages of nerve damage (4,5).In vivo corneal confocal microscopy (IVCCM) is a rapidly expanding technique to quantify the severity of neuropathy in DSPN (6). It has been used to demonstrate early nerve damage in diabetes and a range of other peripheral neuropathies (7,8) with good sensitivity and specificity (9). Recently, corneal nerve damage detected with IVCCM has been related to the level of previous glycemic exposure and blood pressure (10) and HbA_1c even in healthy subjects (11). In a study of subjects with idiopathic small fiber neuropathy, corneal nerve damage was associated with higher serum triglycerides (8). It has also shown significant nerve regeneration before improvement in a range of established measures of neuropathy, including quantitative sensory testing, neurophysiology, and intraepidermal NF density, after simultaneous pancreas and kidney transplantation (12) and after an improvement in glycemia and cardiovascular risk factors for DSPN (13).Corneal NF loss correlates with intraepidermal NF loss (4), and corneal NF length (CNFL), particularly, has shown superior discriminative capacity to diagnose DSPN (14). Recent studies show that quantification of corneal nerve morphology is highly reproducible and does not differ significantly between observers (15) and occasions (16) in subjects with diabetes and healthy individuals. As a functional correlate, corneal sensation has been found to decrease with increasing neuropathic severity (17).Perkins et al. (18) and Bromberg and Jaros (19) have previously reported high interside symmetry of nerve conduction studies (NCS) consistent with the symmetrical nature of diabetic neuropathy. Whilst Petropoulos et al. (16) have shown that central corneal innervation is highly symmetrical between right eyes (REs) and left eyes (LEs) of young healthy subjects, it is unknown whether corneal nerve loss in diabetic neuropathy maintains its symmetry in different stages of DSPN. This is relevant to further establish parallels in terms of pathophysiology between corneal and peripheral somatic nerve damage but also has practical relevance when examining patients to allow examination of only one eye. The purpose of the present, cross-sectional, observational study was to establish if corneal nerve loss, detected using IVCCM, is symmetrical between REs and LEs with increasing severity of diabetic neuropathy.     

Matrix Gla Protein Species and Risk of Cardiovascular Events in Type 2 Diabetic Patients

OBJECTIVE

To investigate the relationship of circulating matrix Gla protein (MGP) species with incident cardiovascular disease (CVD) or coronary heart disease (CHD) in type 2 diabetic patients.

RESEARCH DESIGN AND METHODS

EPIC-NL is a prospective cohort study among 40,011 Dutch men and women. At baseline (1993–1997), 518 participants were known to have type 2 diabetes. MGP levels were measured by ELISA techniques in baseline plasma samples. The incidence of fatal and nonfatal CVD and CVD subtypes—CHD, peripheral arterial disease (PAD), heart failure, and stroke—were obtained by linkage to national registers. Cox proportional hazard models were used to calculate hazard ratios (HRs), adjusted for sex, waist-to-hip ratio, physical activity, and history of CVD.

RESULTS

During a median 11.2 years of follow-up, 160 cases of CVD were documented. Higher circulating desphospho-uncarboxylated MGP (dp-ucMGP) levels were significantly associated with higher risk of CVD, with an HR per SD (HR_SD) of 1.21 (95% CI 1.06–1.38), PAD (HR_SD 1.32 [95% CI 1.07–1.65]), and heart failure (HR_SD 1.75 [95% CI 1.42–2.17]) after adjustment. Higher circulating dp-ucMGP levels were not related to risk of CHD (HR_SD 1.12 [95% CI 0.94–1.34]) or stroke (HR_SD 1.05 [95% CI 0.73–1.49]). Circulating desphospho-carboxylated MGP and circulating total-uncarboxylated MGP levels were not associated with CVD or CVD subtypes.

CONCLUSIONS

High dp-ucMGP levels were associated with increased CVD risk among type 2 diabetic patients, especially with the subtypes PAD and heart failure, while other MGP species were not related to CVD risk. These results suggest that a poor vitamin K status is associated with increased CVD risk.Coronary artery calcification is an independent predictor of cardiovascular disease (CVD) (1). Matrix Gla protein (MGP) is a vitamin K–dependent protein and a potent inhibitor of vascular calcification (2). The importance of MGP for vascular health has been demonstrated in MGP-deficient animals, who all died of massive arterial calcification within 6–8 weeks after birth (3). The cellular and molecular mechanisms by which MGP prevents ectopic calcium deposition are multifaceted, including 1) regulation of calcification by vascular smooth muscle cell (VSMC)-derived matrix vesicles and apoptotic body, 2) inhibition of calcium-phosphate precipitation, and 3) inhibition of VSMC trans differentiation (4). Vitamin K is required for the function of MGP through its role as a cofactor for the enzyme γ-glutamyl carboxylase, catalyzing the carboxylation of glutamic acid residues (Glu) into γ-carboxyglutamate (Gla) at five well-defined places in the protein (5). Human studies showed that high vitamin K intake is associated with reduced coronary artery calcification and reduced risk of CVD (6–9). These effects are thought to be mediated by increased activation of MGP (10).MGP exists as various species, which differ in their state of phosphorylation or carboxylation: phosphorylated, nonphosphorylated (desphospho-MGP [dpMGP]), carboxylated (cMGP), or uncarboxylated (ucMGP). Total uncarboxylated MGP (t-ucMGP) is thought to be the sum of desphospho-uncarboxylated MGP (dp-ucMGP) and phosphorylated-uncarboxylated MGP (p-ucMGP) and mainly consists of p-ucMGP.Development of assays to measure circulating MGP species enabled the investigation of these species in the circulation (11). These studies have shown that dp-ucMGP is a marker for vitamin K status, with high dp-ucMGP level reflecting a low vitamin K status (12–17). In line with these results, several studies indeed showed that high dp-ucMGP levels were associated with more calcification, though not consistently (13,16,18,19). Theoretically, dp-cMGP forms the mirror image of dp-ucMGP and is hypothesized to be associated with lower calcification, but results from human, observational studies are inconsistent (16,20). Finally, in cross-sectional studies, high t-ucMGP has been associated with decreased calcification (16,21–23).Although vascular calcification may not be causally related to CVD, it has emerged as a strong and independent risk marker for CVD (1), but the association of MGP species with CVD events has not been investigated to date. Diabetes is associated with severe cardiovascular complications, including vascular calcification and accelerated atherosclerosis, leading to increased morbidity and mortality in diabetic patients (24–27) Therefore, we performed a prospective study to investigate the association between circulating MGP species and CVD or coronary heart disease (CHD) risk among a high-risk population, i.e., type 2 diabetic patients.     

Inflammation and Cognitive Dysfunction in Type 2 Diabetic Carotid Endarterectomy Patients

OBJECTIVE

Type 2 diabetic patients have a high incidence of cerebrovascular disease, elevated inflammation, and high risk of developing cognitive dysfunction following carotid endarterectomy (CEA). To elucidate the relationship between inflammation and the risk of cognitive dysfunction in type 2 diabetic patients, we aim to determine whether elevated levels of systemic inflammatory markers are associated with cognitive dysfunction 1 day after CEA.

RESEARCH DESIGN AND METHODS

One hundred fifteen type 2 diabetic CEA patients and 156 reference surgical patients were recruited with written informed consent in this single-center cohort study. All patients were evaluated with an extensive battery of neuropsychometric tests. Preoperative monocyte counts, HbA_1c, C-reactive protein (CRP), intercellular adhesion molecule 1, and matrix metalloproteinase 9 activity levels were obtained.

RESULTS

In a multivariate logistic regression model constructed to identify predictors of cognitive dysfunction in type 2 diabetic CEA patients, each unit of monocyte counts (odds ratio [OR] 1.76 [95% CI 1.17–2.93]; P = 0.005) and CRP (OR 1.17 [1.10–1.29]; P < 0.001) was significantly associated with higher odds of developing cognitive dysfunction 1 day after CEA in type 2 diabetic patients.

CONCLUSIONS

Type 2 diabetic patients with elevated levels of preoperative systemic inflammatory markers exhibit more cognitive dysfunction 1 day after CEA. These observations have implications for the preoperative medical management of this high-risk group of surgical patients undergoing carotid revascularization with CEA.The incidence of ischemic stroke is significantly higher in type 2 diabetic patients (1,2), as type 2 diabetes is an independent risk factor for stroke and its recurrence (3,4). Carotid artery stenosis is a major cause of ischemic stroke and can be surgically treated with carotid endarterectomy (CEA). In previous work, we have demonstrated that ∼25% of CEA patients exhibit cognitive dysfunction, a subtle form of neurologic injury, within 1 day of CEA (5,6). Glial markers of neuronal injury (S100B) are elevated in patients who exhibit cognitive dysfunction within 1 day of CEA (7) and reflect opening of the blood–brain barrier (8). Additionally, we have data that demonstrate cognitive dysfunction exhibited within 1 day of CEA is associated with earlier mortality after CEA (9); patients who exhibit cognitive dysfunction within 1 day of CEA experience mortality 4 years earlier than those who do not exhibit cognitive dysfunction within 1 day of CEA. We have also demonstrated that type 2 diabetes is an independent risk factor for cognitive dysfunction (10). In this study, we will investigate factors that might contribute to the increased risk of type 2 diabetic patients undergoing CEA to exhibit the subtle, but significant, cognitive dysfunction.Type 2 diabetes has been associated with accelerated atherosclerosis (11) and elevated systemic inflammation (12–14). Inflammation may play a significant role in accounting for the increased risk of cognitive dysfunction in type 2 diabetic patients. Studies have shown that monocyte activation and infiltration are specifically implicated in the initiation of chronic inflammation and atherosclerosis (12). C-reactive protein (CRP) is a nonspecific marker of systemic inflammation that has been strongly associated with adverse cardiovascular outcomes in both healthy patients and those with coronary artery disease (15–18). Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on endothelial cells and cells of the immune system (19). Matrix metalloproteinase-9 (MMP-9) is secreted from macrophages (20), is involved in the breakdown of vasculature extracellular matrices, and has also been investigated for its various roles in inflammation (21–24). Many previous studies have demonstrated that CRP, ICAM-1, monocytes, and MMP-9 activity are all elevated in type 2 diabetic patients compared with nondiabetic patients. In our previous work, we have demonstrated that type 2 diabetes is a risk factor for cognitive dysfunction (10). We have also previously shown that elevated levels of ICAM-1 (25), monocyte counts (26), and MMP-9 activity (27) are associated with higher incidences of cognitive dysfunction in nondiabetic patients following CEA. We have yet to demonstrate a relationship between CRP and cognitive dysfunction. However, given the previous work done on CRP, we are inspired to do so in this study.Considering previous findings that 1) type 2 diabetes is a risk factor for cognitive dysfunction following CEA; 2) elevated ICAM-1, MMP-9, and monocyte counts are associated with more cognitive dysfunction in CEA patients; and 3) type 2 diabetic patients have elevated levels of CRP, ICAM-1, MMP-9 activity, and monocytes, we hypothesize that type 2 diabetic patients with elevated preoperative systemic inflammation are more likely to exhibit cognitive dysfunction following CEA than those with lower preoperative systemic inflammation. To date, there are no studies that investigate this relationship.We will evaluate preoperative systemic inflammation by measuring CRP, ICAM-1, MMP-9 activity, and monocytes and compare these levels between type 2 diabetic patients with and without cognitive dysfunction 1 day after CEA.     

��-Cell�CMediated Signaling Predominates Over Direct ��-Cell Signaling in the Regulation of Glucagon Secretion in Humans

OBJECTIVE

Given evidence of both indirect and direct signaling, we tested the hypothesis that increased β-cell–mediated signaling of α-cells negates direct α-cell signaling in the regulation of glucagon secretion in humans.

RESEARCH DESIGN AND METHODS

We measured plasma glucagon concentrations before and after ingestion of a formula mixed meal and, on a separate occasion, ingestion of the sulfonylurea glimepiride in 24 basal insulin-infused, demonstrably β-cell–deficient patients with type 1 diabetes and 20 nondiabetic, demonstrably β-cell–sufficient individuals; the latter were infused with glucose to prevent hypoglycemia after glimepiride.

RESULTS

After the mixed meal, plasma glucagon concentrations increased from 22 ± 1 pmol/l (78 ± 4 pg/ml) to 30 ± 2 pmol/l (103 ± 7 pg/ml) in the patients with type 1 diabetes but were unchanged from 27 ± 1 pmol/l (93 ± 3 pg/ml) to 26 ± 1 pmol/l (89 ± 3 pg/ml) in the nondiabetic individuals (P < 0.0001). After glimepiride, plasma glucagon concentrations increased from 24 ± 1 pmol/l (83 ± 4 pg/ml) to 26 ± 1 pmol/l (91 ± 4 pg/ml) in the patients with type 1 diabetes and decreased from 28 ± 1 pmol/l (97 ± 5 pg/ml) to 24 ± 1 pmol/l (82 ± 4 pg/ml) in the nondiabetic individuals (P < 0.0001). Thus, in the presence of both β-cell and α-cell secretory stimuli (increased amino acid and glucose levels, a sulfonylurea) glucagon secretion was prevented when β-cell secretion was sufficient but not when β-cell secretion was deficient.

CONCLUSIONS

These data indicate that, among the array of signals, indirect reciprocal β-cell–mediated signaling predominates over direct α-cell signaling in the regulation of glucagon secretion in humans.The regulation of pancreatic islet α-cell glucagon secretion is complex (1–10). It involves direct signaling of α-cells (1) and indirect signaling of α-cells by β-cell (2–6) and δ-cell (7) secretory products, the autonomic nervous system (8,9), and gut incretins (10).Appropriate glucagon secretory responses occur from the perfused pancreas (3,5) and perifused islets (2). Low plasma glucose concentrations stimulate glucagon secretion from the transplanted (i.e., denervated) human pancreas (11) and the denervated dog pancreas (12). Therefore, we have focused on the intraislet regulation of glucagon secretion. Furthermore, because selective destruction of β-cells results in loss of the glucagon response to hypoglycemia in type 1 diabetes (13), and partial reduction of the β-cell mass in minipigs results in impaired postprandial suppression of glucagon secretion (14), we have focused on the role of β-cell–mediated signaling in the regulation of glucagon secretion.Findings from studies of the perfused rat (3,4) and human (5) pancreas, rats in vivo (6), rat islets (2), isolated rat α-cells (2), and humans (15–18) have been interpreted to indicate that a β-cell secretory product or products tonically restrains basal α-cell glucagon secretion during euglycemia and that a decrease in β-cell secretion, coupled with low glucose concentrations at the α-cells, signals an increase in glucagon secretion in response to hypoglycemia. Parenthetically, the relative roles of the candidate β-cell secretory products (insulin, zinc, γ-aminobutyric acid, and amylin, among others) (2) that normally restrain α-cell glucagon secretion remain to be determined. However, that interpretation rests, in part, on results of studies in isolated rat α-cells (2), which are debated (1), and on the evidence that the islet microcirculation flows from β-cells to α-cells to δ-cells (4), which is also debated (19). Furthermore, it does not address the plausible possibility that a decrease in intraislet δ-cell somatostatin secretion might also signal an increase in α-cell glucagon secretion during hypoglycemia (7).Given that interpretation, it follows that an increase in β-cell secretion would signal a decrease in glucagon secretion in the postprandial state (14). The concept is an interplay of indirect reciprocal β-cell–mediated signaling of α-cells and of direct α-cell signaling in the regulation of glucagon secretion.There is, in our view, compelling evidence that, among other mechanisms, both indirect reciprocal β-cell–mediated signaling of α-cells (2–6) and direct α-cell signaling (1) are involved in the regulation of glucagon secretion by nutrients, hormones, neurotransmitters, and drugs. Given that premise, we posed the question: Which of these predominates in humans? Accordingly, we tested the hypothesis that increased β-cell–mediated signaling of α-cells negates direct α-cell signaling in the regulation of glucagon secretion in humans. To do so, we measured plasma glucagon responses to ingestion of a mixed meal and, on a separate occasion, to ingestion of the sulfonylurea glimepiride in patients with type 1 diabetes and in nondiabetic individuals. We conceptualized patients with type 1 diabetes as a model of α-cells isolated from β-cells because their β-cells had been destroyed but they have functioning α-cells. (Their α-cells are not, of course, isolated from other islet cells, including δ-cells.) Increased plasma amino acid and glucose levels after a mixed meal and sulfonylureas normally stimulate β-cell secretion; increased plasma amino acid and perhaps glucose (2) levels after a mixed meal and sulfonylureas (1) stimulate α-cell secretion. Our hypothesis predicts that such factors that normally stimulate both β-cells and α-cells would stimulate glucagon secretion in patients with type 1 diabetes but not in nondiabetic individuals, i.e., in the virtual absence and the presence of β-cell function, respectively. Indeed, a mixed meal (20,21) and the secretagogues tolbutamide (22), glyburide (23), and repaglinide (23) have been reported to raise plasma glucagon concentrations in patients with type 1 diabetes, but all of those studies lacked nondiabetic control subjects.     

Dipeptidyl peptidase-4 inhibitors and bone fractures: a meta-analysis of randomized clinical trials

OBJECTIVE

Thiazolidinediones and insulin are associated with a higher risk of fractures in type 2 diabetic patients. Incretin hormones increase bone density in experimental models, but the effect of dipeptidyl peptidase-4 (DPP-4) inhibitors on bone fractures has not been reported so far.

RESEARCH DESIGN AND METHODS

A meta-analysis was performed including all randomized clinical trials with a duration of at least 24 weeks, enrolling patients with type 2 diabetes, comparing DPP-4 inhibitors with placebo or active drugs.

RESULTS

Twenty-eight trials enrolling 11,880 and 9,175 patients for DPP-4 inhibitors and comparators, respectively, were included, reporting 63 fractures. DPP-4 inhibitors, compared with placebo or other treatments, were associated with a reduced risk of fractures (Mantel–Haenszel odds ratio [MH-OR] 0.60, 95% CI 0.37–0.99, P = 0.045), even after the exclusion of comparisons with thiazolidinediones or sulfonylureas (MH-OR 0.56, 0.33–0.93, P = 0.026).

CONCLUSIONS

The present meta-analysis suggests that treatment with DPP-4 inhibitors could be associated with a reduced risk of bone fractures.Type 2 diabetes is associated with an increased risk for bone fractures (1–3). The higher risk could be determined by several factors, including falls, diabetes complications, and comorbidities (2). Moreover, glucose-lowering agents such as thiazolidinediones have been reported to reduce bone density (4,5) and to increase the incidence of fractures in longer-term trials (6,7) and in epidemiologic studies (8). Insulin therapy is also associated with an increased fracture risk (9–11) despite its neutral effect on bone density (12). The increased risk of falls, due to hypoglycemia, could lead to higher fracture risk (10).Glucagon-like peptide-1 (GLP-1) has been reported to induce osteoblast differentiation (13) and inhibit osteoclastic activity (14); GLP-1 receptor agonists stimulate bone formation in rodents (15). Experimental data in animal models suggest that gastric intestinal polypeptide is also capable of increasing bone density (16,17). Drugs capable of increasing incretin levels, such as dipeptidyl peptidase-4 (DPP-4) inhibitors, could therefore exert beneficial effects on the bone.