A data-driven architecture using natural language processing to improve phenotyping efficiency and accelerate genetic diagnoses of rare disorders.

Effective genetic diagnosis requires the correlation of genetic variant data with detailed phenotypic information. However, manual encoding of clinical data into machine-readable forms is laborious and subject to observer bias. Natural language processing (NLP) of electronic health records has great potential to enhance reproducibility at scale but suffers from idiosyncrasies in physician notes and other medical records. We developed methods to optimize NLP outputs for automated diagnosis. We filtered NLP-extracted Human Phenotype Ontology (HPO) terms to more closely resemble manually extracted terms and identified filter parameters across a three-dimensional space for optimal gene prioritization. We then developed a tiered pipeline that reduces manual effort by prioritizing smaller subsets of genes to consider for genetic diagnosis. Our filtering pipeline enabled NLP-based extraction of HPO terms to serve as a sufficient replacement for manual extraction in 92% of prospectively evaluated cases. In 75% of cases, the correct causal gene was ranked higher with our applied filters than without any filters. We describe a framework that can maximize the utility of NLP-based phenotype extraction for gene prioritization and diagnosis. The framework is implemented within a cloud-based modular architecture that can be deployed across health and research institutions.

Diagnosis of genetic diseases in seriously ill children by rapid whole-genome sequencing and automated phenotyping and interpretation.

By informing timely targeted treatments, rapid whole-genome sequencing can improve the outcomes of seriously ill children with genetic diseases, particularly infants in neonatal and pediatric intensive care units (ICUs). The need for highly qualified professionals to decipher results, however, precludes widespread implementation. We describe a platform for population-scale, provisional diagnosis of genetic diseases with automated phenotyping and interpretation. Genome sequencing was expedited by bead-based genome library preparation directly from blood samples and sequencing of paired 100-nt reads in 15.5 hours. Clinical natural language processing (CNLP) automatically extracted children's deep phenomes from electronic health records with 80% precision and 93% recall. In 101 children with 105 genetic diseases, a mean of 4.3 CNLP-extracted phenotypic features matched the expected phenotypic features of those diseases, compared with a match of 0.9 phenotypic features used in manual interpretation. We automated provisional diagnosis by combining the ranking of the similarity of a patient's CNLP phenome with respect to the expected phenotypic features of all genetic diseases, together with the ranking of the pathogenicity of all of the patient's genomic variants. Automated, retrospective diagnoses concurred well with expert manual interpretation (97% recall and 99% precision in 95 children with 97 genetic diseases). Prospectively, our platform correctly diagnosed three of seven seriously ill ICU infants (100% precision and recall) with a mean time saving of 22:19 hours. In each case, the diagnosis affected treatment. Genome sequencing with automated phenotyping and interpretation in a median of 20:10 hours may increase adoption in ICUs and, thereby, timely implementation of precise treatments.

A Bayesian mathematical model of motor and cognitive outcomes in Parkinson's disease.

BACKGROUND: There are few established predictors of the clinical course of PD. Prognostic markers would be useful for clinical care and research. OBJECTIVE: To identify predictors of long-term motor and cognitive outcomes and rate of progression in PD. METHODS: Newly diagnosed PD participants were followed for 7 years in a prospective study, conducted at 55 centers in the United States and Canada. Analyses were conducted in 244 participants with complete demographic, clinical, genetic, and dopamine transporter imaging data. Machine learning dynamic Bayesian graphical models were used to identify and simulate predictors and outcomes. The outcomes rate of cognition changes are assessed by the Montreal Cognitive Assessment scores, and rate of motor changes are assessed by UPDRS part-III. RESULTS: The most robust and consistent longitudinal predictors of cognitive function included older age, baseline Unified Parkinson's Disease Rating Scale (UPDRS) parts I and II, Schwab and England activities of daily living scale, striatal dopamine transporter binding, and SNP rs11724635 in the gene BST1. The most consistent predictor of UPDRS part III was baseline level of activities of daily living (part II). Key findings were replicated using long-term data from an independent cohort study. CONCLUSIONS: Baseline function near the time of Parkinson's disease diagnosis, as measured by activities of daily living, is a consistent predictor of long-term motor and cognitive outcomes. Additional predictors identified may further characterize the expected course of Parkinson's disease and suggest mechanisms underlying disease progression. The prognostic model developed in this study can be used to simulate the effects of the prognostic variables on motor and cognitive outcomes, and can be replicated and refined with data from independent longitudinal studies.

Bayesian Network Inference Modeling Identifies TRIB1 as a Novel Regulator of Cell-Cycle Progression and Survival in Cancer Cells.

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G1-S transition. The model identified known components of the cell-cycle machinery, such as CCND1, CCNE2, and CDC25A, as well as revealed novel regulators of G1-S transition, IER2, TRIB1, TRIM27. Experimental validation of model predictions confirmed 10 of 12 predicted genes to have a role in G1-S progression. Further analysis showed that TRIB1 regulated the cyclin D1 promoter via NFκB and AP-1 sites and sensitized cells to TRAIL-induced apoptosis. In clinical specimens of breast cancer, TRIB1 levels correlated with expression of NFκB and its target genes (IL8, CSF2), and TRIB1 copy number and expression were predictive of clinical outcome. Together, our results establish a critical role of TRIB1 in cell cycle and survival that is mediated via the modulation of NFκB signaling. Cancer Res; 77(7); 1575-85. ©2017 AACR.

Curcuminoids limit neutrophil-mediated reperfusion injury in experimental stroke by targeting the endothelium.

OBJECTIVE: We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion. METHODS: Male Sprague Dawley rats subjected to MCAO/R were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema, and neurological function were assessed at 24 hours. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed. RESULTS: Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC. CONCLUSION: Turmeric-derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.

Acute cardiovascular effects of firefighting and active cooling during rehabilitation.

OBJECTIVES: To determine the cardiovascular and hemostatic effects of fire suppression and postexposure active cooling. METHODS: Forty-four firefighters were evaluated before and after a 12-minute live-fire drill. Next, 50 firefighters performing the same drill were randomized to undergo postfire forearm immersion in 10 °C water or standard rehabilitation. RESULTS: In the first study, heart rate and core body temperature increased and serum C-reactive protein decreased but there were no significant changes in fibrinogen, sE-selectin, or sL-selectin. The second study demonstrated an increase in blood coagulability, leukocyte count, factors VIII and X, cortisol, and glucose, and a decrease in plasminogen and sP-selectin. Active cooling reduced mean core temperature, heart rate, and leukocyte count. CONCLUSIONS: Live-fire exposure increased core temperature, heart rate, coagulability, and leukocyte count; all except coagulability were reduced by active cooling.

Intracellular signaling controls endothelial cell prostacyclin secretion and regulation of blood clotting time.

Blood is constantly in contact with a biological material, the blood vessel wall, without the need for anticoagulants to prevent clot formation on the vessel wall; however, man-made biomaterials require anticoagulants to prevent clot formation on the biomaterial. This study seeks to understand how some biomaterials elicit anticoagulant responses from endothelial cells (ECs), whereas others do not. Partial least squares regression analysis was used to correlate the activity of four relevant signaling molecules [extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), Akt, and IκB kinase (IKK)] with human umbilical vein EC secretion of prostacyclin and clotting time of whole blood in contact with these cells. Prostacyclin secretion was increased when JNK activity (mean of all time-points) was elevated and IKK activity at 30 min was reduced. In addition, the clotting time, R-time measured by thromboelastography, was increased (reduced coagulability) when activity of both ERK and JNK (mean of all time-points) were increased and when Akt activity was increased at longer contact times (24-72 h after cell contact with material). Inhibition of each signaling molecule with subsequent testing for prostacyclin secretion and R-time confirmed the interrelationship between EC intracellular signaling and prostacyclin secretion. Generally, JNK inhibition decreased and IKK inhibition increased prostacyclin secretion. Inhibition of ERK or JNK generally increased coagulability, and Akt inhibition decreased the R-time of samples normally eliciting reduced coagulability. These findings increase our understanding of the signaling pathways involved in endothelial prostacyclin release and suggest targets for developing EC-seeded biomaterial surfaces that can minimize coagulation.

Standardized methods to quantify thrombogenicity of blood-contacting materials via thromboelastography.

Blood coagulation is the most significant complication of vascular biomaterials. A straightforward, sensitive, and standard measure of the compatibility of these materials with whole blood (hemocompatibility) is necessary to avoid coagulation. Current techniques used quantify only individual clotting components and are poor predictors of coagulation. The thromboelastograph (TEG) provides a measure of overall clot formation from whole blood. Although TEG is very common in clinical settings, its application to biomaterials is limited partly due to difficulty in sample preparation. In this protocol, whole blood samples are incubated with (1) biomaterials (tube with clamped ends) and (2) endothelial cells cultured on biomaterial surfaces (12-well plate) under controlled shearing conditions (10 rpm on rocker, at 37°C), and then the blood is transferred to the TEG to measure clot formation. TEG clearly discriminates among the R-times (time until initial clot formation) of expanded poly(tetrafluoroethylene), poly(urethane), and Tygon tubing. Marked differences in R-time are also seen when endothelial cells are cultured on various extracellular matrix proteins and proteoglycans. Thus, R-time provides a robust metric of overall thrombogenicity of biomaterials, and these procedures provide a standardized method for TEG to facilitate direct comparison among candidate biomaterials undergoing in-vitro testing.

Exaggerated neutrophil-mediated reperfusion injury after ischemic stroke in a rodent model of type 2 diabetes.

OBJECTIVE: We tested the hypothesis that both chronic and acute inflammatory processes contribute to worse reperfusion injury and stroke outcome in an experimental model of T2DM. MATERIALS AND METHODS: Twelve- to thirteen-week-old male Zucker Diabetic Fatty (ZDF) rats vs. Zucker Lean Controls (ZLC) rats were tested at baseline and after middle cerebral artery occlusion (ischemia) and reperfusion (I-R). Neutrophil adhesion to the cerebral microcirculation, neutrophil expression of CD11b, infarction size, edema, neurologic function, sICAM, and cerebral expression of neutrophil-endothelial inflammatory genes were measured. RESULTS: At baseline, CD11b and sICAM were significantly increased in ZDF vs. ZLC animals (p < 0.05). After I-R, significantly more neutrophil adhesion and cell aggregates were observed in ZDF vs. ZLC (p < 0.05); infarction size, edema, and neurologic function were significantly worse in ZDF vs. ZLC (p < 0.05). CD11b and sICAM-1 remained significantly increased in ZDFs (p < 0.05), and cerebral expression of IL-1ß, GRO/KC, E-selectin, and sICAM were significantly induced in ZDF, but not ZLC groups (p < 0.05) after 2.5 hours of reperfusion. CONCLUSION: Both sides of the neutrophil-endothelial interface appear to be primed prior to I-R, and remain significantly more activated during I-R in an experimental model of T2DM. Consequently, reperfusion injury appears to play a significant role in poor stroke outcome in T2DM.

Causal modeling using network ensemble simulations of genetic and gene expression data predicts genes involved in rheumatoid arthritis.

Tumor necrosis factor α (TNF-α) is a key regulator of inflammation and rheumatoid arthritis (RA). TNF-α blocker therapies can be very effective for a substantial number of patients, but fail to work in one third of patients who show no or minimal response. It is therefore necessary to discover new molecular intervention points involved in TNF-α blocker treatment of rheumatoid arthritis patients. We describe a data analysis strategy for predicting gene expression measures that are critical for rheumatoid arthritis using a combination of comprehensive genotyping, whole blood gene expression profiles and the component clinical measures of the arthritis Disease Activity Score 28 (DAS28) score. Two separate network ensembles, each comprised of 1024 networks, were built from molecular measures from subjects before and 14 weeks after treatment with TNF-α blocker. The network ensemble built from pre-treated data captures TNF-α dependent mechanistic information, while the ensemble built from data collected under TNF-α blocker treatment captures TNF-α independent mechanisms. In silico simulations of targeted, personalized perturbations of gene expression measures from both network ensembles identify transcripts in three broad categories. Firstly, 22 transcripts are identified to have new roles in modulating the DAS28 score; secondly, there are 6 transcripts that could be alternative targets to TNF-α blocker therapies, including CD86--a component of the signaling axis targeted by Abatacept (CTLA4-Ig), and finally, 59 transcripts that are predicted to modulate the count of tender or swollen joints but not sufficiently enough to have a significant impact on DAS28.

The contribution of mannose binding lectin to reperfusion injury after ischemic stroke.

After complement system (CS) activation, the sequential production of complement products increases cell injury and death through opsonophagocytosis, cytolysis, adaptive, and inflammatory cell responses. These responses potentiate cerebral ischemia-reperfusion (IR) injury after ischemic stroke and reperfusion. Activation of the CS via mannose binding lectin (MBL)-initiated lectin pathway is known to increase tissue damage in response to IR in muscle, myocardium and intestine tissue. In contrast, the contribution of this pathway to cerebral IR injury, a neutrophil-mediated event, is less clear. Therefore, we investigated the potential protective role of MBL deficiency in neutrophil-mediated cerebral injury after IR. Using an intraluminal filament method, neutrophil activation and cerebral injury were compared between MBL-deficient and wild type C57Bl/6 mice subjected to 60 minutes of MCA ischemia and reperfusion. Systemic neutrophil activation was not decreased in MBL-deficient animals after IR. In MBL-deficient animals, cerebral injury was significantly decreased only in the striatum (p < 0.05). Despite MBL deficiency, C3 depositions were evident in the injured hemisphere during reperfusion. These results indicate that while MBL deficiency results in a modest protection of a sub-cortical brain region during IR, redundant complement pathway activation may overwhelm further beneficial effects of MBL deficiency during reperfusion.

Clinical determinants of aspirin resistance in diabetes.

Recent studies indicate that not all diabetic subjects benefit from aspirin therapy. Our objective is to characterize diabetic subjects with aspirin resistance using urine thromboxane, and VerifyNow measures. Our results suggest that cardiovascular disease, microalbuminuria, poor diabetes control, and increased waist circumference help identify aspirin resistance in diabetes.

Effect of glucose or fat challenge on aspirin resistance in diabetes.

Aspirin has lower antiplatelet activity in diabetic patients. Our aim is to study the roles of acute hyperglycemia and hyperlipidemia on aspirin function in diabetic subjects with and without cardiovascular disease. Using urine thromboxane (pg/mg creatinine) and VerifyNow (Aspirin Resistance Measures-ARU), we investigated diabetic subjects during a 2-hour glucose challenge (n = 49) or a 4-hour fat challenge (n = 11). All subjects were currently taking aspirin (81 or 325 mg). After fat ingestion, urine thromboxane increased in all subjects (Mean ± SE before: after) (1209 ± 336: 1552 ± 371, P = .01), while we noted a trend increase in VerifyNow measures (408 ± 8: 431 ± 18, P = .1). The response to glucose ingestion was variable. Diabetic subjects with cardiac disease and dyslipidemia increased thromboxane (1693 ± 364: 2799 ± 513, P < .05) and VerifyNow (457.6 ± 22.3: 527.1 ± 25.8, P < .05) measures after glucose. We conclude that saturated fat ingestion increases in vivo thromboxane production despite aspirin therapy.

Whole blood aggregation, coagulation, and markers of platelet activation in diet-induced diabetic C57BL/6J mice.

AIMS: Type 2 diabetes in humans is associated with hypercoaguability; however, little is known about platelet function in mouse models of type 2 diabetes used to study this disorder. Therefore, the purpose of this study was to examine platelet aggregation, coagulation, and markers of platelet activation in a diet-induced mouse model of type 2 diabetes. METHODS: Four week old, male, C57BL/6J mice were randomized to a standard chow or high fat (60% beef lard) diet for 4 months. To examine platelet function we measured ADP-induced whole blood aggregometry, whole blood coagulation by thromboelastography, tail bleeding times, platelet microparticle and platelet expression of p-selectin and platelet expression of CD61 by flow cytometry. RESULTS: Diabetic mice exhibited less aggregation (p<0.05), less coagulation (p<0.01), prolonged tail bleeding times (n.s.), and lower agonist stimulated platelet CD61 expression (p<0.001) compared to non-diabetic mice. There was no difference in platelet microparticle and platelet p-selectin expression. CONCLUSIONS: Diet-induced type 2 diabetic mouse do not demonstrate the hypercoagulability and platelet activation typically observed in humans with this disorder. More studies are warranted to further explore platelet function in this mouse model; to determine their applicability for studying these alterations in type 2 diabetes.

Inhibition of platelet GPIIb-IIIa and P-selectin expression by aspirin is impaired by stress hyperglycemia.

Increased aspirin resistance may contribute to the increase in thrombotic events observed in patients with type 2 diabetes. In this study, we examined if acute exposure to increased plasma glucose impaired the inhibitory effects of aspirin on platelet activation. Whole-blood samples were incubated with 100 (euglycemia), 200, 300, and 600 mg/dl glucose followed by incubation with aspirin [acetylsalicylic acid (ASA)]. Using flow cytometry, GPIIb-IIIa and P-selectin were analyzed in unstimulated and arachidonic acid (AA)-stimulated platelets. In euglycemic blood, AA caused a significant increase in platelet GPIIb-IIIa expression [unstimulated: 59.5+/-8.2 total fluorescence intensity (TFI), AA stimulated: 319.6+/-42.7 TFI, P=.002] and P-selectin (4.4+/-0.7 and 179.5+/-38.5 TFI, P<.001). In vitro, ASA significantly inhibited both GPIIb-IIIa expression (36.5%) and P-selectin expression (81%; P<.005). However, increased blood glucose (200 mg/dl) significantly impaired the inhibitory effect of ASA (84% for GPIIb-IIIa, P<.005; 48% for P-selectin, P=NS). Increasing glucose to 600 mg/dl completely overwhelmed the inhibitory effect of ASA. A statistically significant interaction between glucose concentration and ASA dose was found (P<.001 for GPIIb-IIIa and P=.004 for P-selectin). In vitro, concentration-dependent stress hyperglycemia significantly impaired the inhibitory effects of aspirin on human platelet GPIIb-IIIa and P-selectin expression. Under acute hyperglycemic conditions, the effectiveness of ASA to inhibit platelets via the AA-activation pathway may be significantly reduced.

The mannose-binding lectin pathway is a significant contributor to reperfusion injury in the type 2 diabetic heart.

The severity of ischaemic heart disease is markedly enhanced in type 2 diabetes. We recently reported that complement activation exacerbates I/R injury in the type 2 diabetic heart. The purpose of this study was to isolate and examine MBL pathway activation following I/R injury in the diabetic heart. ZLC and ZDF rats underwent 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Two different groups of ZDF rats were treated with either FUT-175, a broad complement inhibitor, or P2D5, a monoclonal antibody raised against rat MBL-A. ZDF rats treated with FUT175 and P2D5 had significantly decreased myocardial infarct size, C3 deposition and neutrophil accumulation compared with untreated ZDF controls. Taken together, these findings indicate that the MBL pathway plays a key role in the severity of complement-mediated I/R injury in the type 2 diabetic heart.

Caspase inhibition of platelet activation.

The role of caspases in platelet function is not well understood. When platelets are activated, they express phosphatidylserine on the outer plasma membrane, form platelet microparticles, and aggregate (Pag). The aims of this study were to determine if caspases play a role in the platelet activation seen in type 2 diabetes. Diabetic rats (Zucker diabetic fatty) were treated with a broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone, in vivo and platelets were evaluated for phosphatidylserine expression, platelet microparticle formation, and Pag. We found a decreased phosphatidylserine exposure in zVAD-Zucker diabetic fatty rats compared to Zucker diabetic fatty-phosphate-buffered saline when activated with 20 micromol/l ADP. Zucker diabetic fatty rats treated with benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone decreased platelet microparticle numbers compared to phosphate-buffered saline control Zucker diabetic fatty rats. Further, treatment with benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone significantly decreased Pag. These results indicate that caspases play a role in platelet activation, suggesting a unique physiologic role of these proteases and perhaps the underlying mechanisms involved in the chronic platelet activation observed in type 2 diabetes.

Inflammatory and hemodynamic changes in the cerebral microcirculation of aged rats after global cerebral ischemia and reperfusion.

Effects of aging on inflammation and blood flow in the brain are unclear. Young (three to six months) and aged (19-22 months) male Brown Norway Fisher rats were used to compare (i) leukocyte function in nonischemic conditions and (ii) leukocyte function and hemodynamic changes after ischemia-reperfusion (I-R). In nonischemic studies, polymorphonuclear (PMN) CD11b expression and reactive oxygen species (ROS) production were measured with flow cytometry and PMN chemotaxis was measured with a Boyden chamber (+/-fMLP). In I-R studies, ischemia was induced by bilateral carotid artery occlusion and hypotension (20 minutes). During early reperfusion (30 minutes), leukocyte adhesion and rolling and blood-shear rates were measured using fluorescence microscopy. During late reperfusion (48 hours), mortality, neurological function, and leukocyte infiltration were measured. Stimulated PMN chemotaxis was increased in nonischemic aged rats (p < 0.05). In early reperfusion, there was a significant increase in leukocyte rolling and adhesion in the cerebral microcirculation and a significant decrease in shear rate in aged rats, compared to the young (p < 0.05). During late reperfusion, neurologic function was worse in aged vs. young rats (p < 0.05). These findings suggest that increased intravascular PMN adhesion and vascular dysfunction may contribute to poor neurologic outcome after cerebral I-R in the aged brain.

Whole blood aggregation and coagulation in db/db and ob/ob mouse models of type 2 diabetes.

Type 2 diabetes in humans is associated with a significant hypercoagulable state; however, the effects of this on stroke and cardiovascular disease are not completely understood. The genetic mutations in db/db and ob/ob mice produce metabolic abnormalities similar to type 2 diabetes, but little is known about their platelet or coagulation properties. The objective of this study was therefore to examine platelet function and coagulation in db/db and ob/ob mice to determine the degree of alteration induced by type 2 diabetes. Male db/db and ob/ob mice, 8-16 weeks old, and their respective genetic control mice were used for all experiments. To examine platelet function and coagulation, we measured ADP-induced whole blood aggregation at baseline and after inhibition with aspirin and fucoidan, whole blood coagulation by thromboelastography, and platelet CD61 expression by flow cytometry. Both db/db and ob/ob mice demonstrated significantly less ADP-induced whole blood aggregation compared with control mice (db/db mice, P < 0.001; ob/ob mice, P < 0.01). Aggregation was significantly inhibited with aspirin in all groups; however, fucoidan inhibited aggregation only in control mice. The db/db and ob/ob mice demonstrated significantly less maximal clot strength compared with control mice (P < 0.01), and ob/ob mice demonstrated premature clot fibrinolysis measured by thromboelastography. In conclusion, the db/db and ob/ob type 2 diabetes mouse models do not demonstrate a hypercoagulable state similar to humans with this disease. We caution their use for studying cardiovascular and cerebrovascular disease in the setting of type 2 diabetes.

Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion.

Chronic inflammation exacerbates the cardiovascular complications of diabetes. Complement activation plays an important role in the inflammatory response and is known to be involved in ischemia-reperfusion (I/R) injury in the nondiabetic heart. The purpose of this study was to determine if increased complement deposition explains, in part, the increased severity of neutrophil-mediated I/R injury in the type 2 diabetic heart. Nondiabetic Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats underwent 30 min of coronary artery occlusion followed by 120 min of reperfusion. Another group of ZDF rats was treated with the complement inhibitor FUT-175 before reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition and neutrophil accumulation following reperfusion. We found significantly more complement deposition in the ZDF LV compared with the ZLC (P < 0.05), and complement deposition was associated with significantly greater neutrophil accumulation. In whole blood samples taken preischemia and at 120 min reperfusion, neutrophils exhibited significantly more CD11b expression in the ZDF group compared with the ZLC group (P < 0.05). Furthermore, intracellular adhesion molecule (ICAM)-1 expression following I/R was increased significantly in ZDF hearts compared with ZLC hearts (P < 0.001). These results indicate that, in the ZDF heart, increased ICAM-1 and polymorphonuclear neutrophil (PMN) CD11b expression play a role in increasing PMN accumulation following I/R. The infarct size of the ZDF was significantly greater than ZLC (P < 0.05), and treatment with FUT-175 significantly decreased infarct size, complement deposition, and PMN accumulation in the diabetic heart. These findings indicate an exacerbated inflammatory response in the type 2 diabetic heart that contributes to the increased tissue injury observed following ischemia and reperfusion.