IKKß regulates endothelial thrombomodulin in a Klf2-dependent manner.

BACKGROUND: Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNFα) suppresses TM expression. OBJECTIVE: TNFα has been shown to down-regulate TM partly via activation of nuclear factor kappa B (NF-κB). However, because the TM promoter lacks an NF-κB binding site, the direct involvement of NF-κB has been controversial. We investigated the role of the upstream regulatory serine kinase, inhibitory kappa-B kinase-ß (IKKß), in TM expression and function with or without TNFα treatment. METHODS: Inhibition of IKKß was achieved by specific chemical inhibitors, siRNA or shRNA. TM expression was assessed by qRT-PCR, Western blot, flow cytometry, luciferase reporter assay and chromatin immune-precipitation (ChIP) assay. TM function was estimated by generation of activated protein C (APC). NF-κB activation was determined by immunocytochemistry. RESULTS AND CONCLUSIONS: IKKß inhibition increased TM expression and function, and attenuated TNFα-mediated TM down-regulation. In contrast, inhibition of downstream canonical NF-κB protein family members p50 and p65 (RelA) failed to up-regulate TM expression and did not affect IKKß inhibition-mediated TM over-expression. However, knockdown of cRel and RelB, family members of the canonical and non-canonical NF-κB pathway, respectively, resulted in TM over-expression. IKKß inhibition caused over-expression, increased promoter activity and enhanced binding of Krüppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKKß inhibition-mediated TM up-regulation. We conclude that IKKß regulates TM in a Klf2-dependent manner.

Influence of statins on postoperative wound complications after inguinal or ventral herniorrhaphy.

The lipid-lowering agents, statins, are the most commonly prescribed class of drugs in the western world. Because of their widespread use, many patients undergo surgical procedures while on statins. Statins, in addition to cholesterol-lowering effects, also have anticoagulant, immunosuppressive, and antiproliferative properties that may affect the risk of local wound complications. This study investigated the relationship between statins and postoperative wound complications in a large cohort of patients undergoing inguinal or ventral hernia repair. Data mining was performed in the Veterans Integrated Service Network (VISN)16 Data Warehouse. This database contains clinical and demographic information about all veterans cared for at the ten VA Medical Centers that comprise the South Central VA Healthcare Network in the mid-south region of the US. Aggregate data (age, body mass index, smoking history, gender, race, history of diabetes, statin use, and postoperative wound complications) were obtained for all patients who underwent inguinal or ventral hernia repair during the period October 1, 1996-November 30, 2004. During the period of the query, 10,782 patients (10,676 male, 106 female), 1,242 (11.5%) of whom received statins, underwent herniorrhaphy. Statin use did not affect the risk of wound infection or delayed wound healing. Statin use was, however, associated with an increased rate of local postoperative bleeding complications (P=0.01). When the type of hernia, age, smoking, diabetes, and body mass index were included in a multivariate analysis, statins remained borderline significant as an independent predictor of wound hematoma/postoperative bleeding (P=0.04), odds ratio 1.6 (95% CI 1.03-2.44). Patients who undergo inguinal herniorrhaphy while on statins have an increased risk of postoperative wound hematoma/hemorrhage. Focus on additional factors that may affect the propensity to postoperative bleeding and on meticulous intraoperative hemostasis are particularly important in such patients.

Hirudin ameliorates intestinal radiation toxicity in the rat: support for thrombin inhibition as strategy to minimize side-effects after radiation therapy and as countermeasure against radiation exposure.

BACKGROUND: The small bowel is a dose-limiting normal tissue in radiation therapy of malignancies in the abdomen and pelvis, as well as an important determinant of survival after non-therapeutic radiation exposure. Irradiation of normal tissues, including intestine, causes loss of vascular thromboresistance and upregulation of thrombin receptors. Radiation-induced endothelial dysfunction is thought to be involved in both early and delayed radiation responses. Hence, thrombin may be a potential target for ameliorating normal tissue radiation toxicity. OBJECTIVE: To assess direct thrombin inhibition as a protective strategy against small bowel radiation toxicity. METHODS: Rat small intestine was exposed to localized orthovoltage X-radiation. Recombinant hirudin, a direct thrombin inhibitor, or vehicle was infused from 2 days before irradiation to 14 days after irradiation. Structural, cellular, and molecular aspects of intestinal radiation injury were assessed at 2 weeks (early toxicity) and 26 weeks (chronic toxicity) after irradiation. RESULTS: Compared with unirradiated intestine, irradiated intestine showed increased expression of tissue factor, increased immunoreactivity for enzymatically active thrombin, and increased extravascular fibrin(ogen) deposition. Hirudin treatment significantly attenuated radiation-induced mucosal damage (P = 0.04), reactive intestinal wall thickening (P = 0.02), transforming growth factor-beta immunoreactivity levels (P = 0.0002), and collagen III deposition (P = 0.003). The differences between hirudin-treated and control rats were more pronounced at 2 weeks than at 26 weeks after irradiation. Hirudin treatment did not affect postradiation granulocyte infiltration. CONCLUSIONS: Short-term thrombin inhibition attenuates important aspects of intestinal radiation toxicity. Thrombin is a promising target for minimizing normal tissue injury after radiation therapy of cancer, as well as for protecting normal tissues from the adverse effects of non-therapeutic radiation exposure.

Advanced prostate cancer activates coagulation: a controlled study of activation markers of coagulation in ambulatory patients with localized and advanced prostate cancer.

Cancer and increased age are risk factors for coagulation activation. Patients with advanced prostate cancer, which usually presents in the seventh to eighth decade of life, are likely to be at increased risk for thrombosis. We report results of a controlled study of changes in specific and sensitive markers of coagulation activation in patients with prostate cancer. Complete blood count, prothrombin time, partial thromboplastin time, prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin complex (TAT) and quantitative D-dimers (DD) were measured in 30 patients of advanced prostate cancer (androgen ablated), in 30 newly diagnosed localized prostate cancer patients, in 30 healthy age-matched volunteers, and in 20 healthy young volunteers. Plasma F1 + 2 (P < 0.05) and DD (P < 0.05), but not TAT, were significantly elevated in healthy elderly males (mean age, 77 years) when compared with healthy young volunteers (mean age, 35 years). F1 + 2, TAT and DD were significantly elevated in advanced prostate cancer when compared with healthy age-matched controls (P < 0.001). In conclusion, advanced prostate cancer patients have significantly increased levels of sensitive markers of coagulation activation compared with healthy age-matched controls. This data can be used to plan studies to determine the risk of clinically significant coagulopathy and the role of primary prophylaxis in patients with advanced prostate cancer.

Cytokines in coagulation and thrombosis: a preclinical and clinical review.

The cytokine network is a complex and dynamic system, involved in numerous biological responses in the human body. This review of the current literature describes the role of cytokines and their interaction with the coagulation system, specifically in the maintenance of the thrombo-hemorrhagic balance in vivo in human subjects and in animals. In general, cytokines are thrombogenic, but they are amenable to therapeutic manipulations and hence are a potentially attractive tool in the clinician's armamentarium. Studies of the effects of cytokines in vivo are difficult because cytokines act in a very finite microenvironment and, although their actions are significant, they are transient. Most of the available clinical data related to interactions between cytokines and the coagulation system focuses on the role of tumor necrosis factor-alpha and interleukin-1 in septicemia and septic shock. However, several other cytokines and related proteins, such as platelet activating factor and plasminogen activator inhibitor, are also known to influence coagulation and thrombosis. These factors interact closely with cytokines, and have been included in this review for a better understanding of their interactions with traditional cytokines. Studies that utilize cell culture systems do not accurately model the in vivo status of this complex system and, hence, this review has excluded such studies. The role of the cytokine network in coronary artery disease, angiogenesis, or neoplasia has been addressed elsewhere by other workers and is not discussed here. By emphasizing important in vivo interactions, the intention of this review is to serve as an impetus to further translational research, both clinical and in the laboratory.

Association between decreased pulmonary endothelial cell thrombomodulin and local fibrin deposition in pneumonia.

Thrombomodulin (TM) plays an important role in anticoagulation by forming a complex with thrombin, which subsequently activates protein C. TM is inactivated and downregulated by inflammatory cell mediators. This study examined whether bronchopneumonia is associated with changes in TM immunoreactivity, and whether a decrease in TM is accompanied by evidence of hypercoagulability, i.e. local deposition of fibrin. Double antibody staining for TM and fibrin was performed on lung tissue sections from patients who had died of pneumonia and from patients who had died rapidly, secondary to trauma. Inflammatory changes were assessed histologically and immunohistochemically using antibodies against interleukin-1alpha, tumor necrosis factor-alpha, and myeloperoxidase. Areas with bronchopneumonia exhibited markedly decreased endothelial TM staining of alveolar walls and small vessels. These changes were associated with prominent fibrin immunoreactivity. Some areas exhibited mild to moderate inflammation with little fibrin deposition and variable amounts of TM in adjacent vessels. This study is the first to relate changes of TM immunoreactivity levels to fibrin deposition in a human disease process. These data may have implications for pulmonary pathophysiology in patients with bronchopneumonia.

The effect of glucose and insulin on the activity of methylene tetrahydrofolate reductase and cystathionine-beta-synthase: studies in hepatocytes.

Hyperhomocysteinemia is a well established risk factor for cardiovascular disease, and multiple factors likely lead to abnormal regulation of plasma homocysteine in patients with diabetes. To examine a possible role for insulin and glucose in homocysteine metabolism, we examined the activity of two important enzymes of homocysteine metabolism in hepatocytes. In various tissues of six mice, methylene tetrahydrofolate reductase (MTHFR) activity was present in all tissues tested and the highest concentration (per gram) was in the brain. In contrast, cystathionine beta-synthase (CBS) activity appeared to be present only in the liver and to a small extent in the kidney. Using HEP G2 cells in culture, MTHFR activity was 3.3+/-0.8 nmol/h when the glucose concentration in the medium was 100 mg/dl and fell to 2.3+/-0.3 nmol/h when glucose was increased to 300 mg/dl. MTHFR activity was 3.4+/-0.3 nmol/h when cells were exposed to an insulin concentration of 5 mU/ml and fell to 2.8+/-0.3 nmol/h when insulin concentration was increased to 200 mU/ml (P<0.01). In contrast CBS activity increased from 0.017 to 0.13 U/ml by increasing the glucose concentration in the medium (P<0.01), but decreased from 0.04 to 0.02 (P<0.01) when the insulin concentration was increased from 5 to 200 mU/ml, respectively. We conclude that CBS and MTHFR have different tissue distributions, with CBS being present predominantly in liver and kidney, and MTHFR found in many tissues. In addition, both insulin and glucose affect the activity of the two enzymes when added to hepatocytes in vitro. If such effects occur in humans with hyperglycemia and hyperinsulinemia, then alterations in homocysteine metabolism may contribute to the accelerated macrovascular disease associated with insulin resistance or type 2 diabetes.

Blood S-adenosylmethionine concentrations and lymphocyte methylenetetrahydrofolate reductase activity in diabetes mellitus and diabetic nephropathy.

The erythrocyte concentrations of the body's chief physiologic methyl donor S-adenosylmethionine (SAM) and of its metabolite and inhibitor S-adenosylhomocysteine (SAH), the plasma concentrations of total homocysteine (tHcy), and the activity of N(5,10) methylenetetrahydrofolate reductase (MTHFR) in lymphocytes were determined in healthy subjects and patients with diabetes mellitus without complications and at various stages of diabetic nephropathy, categorized according to the degree of progression of the disease. These groups were as follows: 1, control; 2, diabetics with no complications; 3, patients with albuminuria; 4, patients with an elevated plasma creatinine; and 5, patients on dialysis. No parameter studied exhibited significant differences between the type 1 and the type 2 diabetics. In control subjects, the blood concentrations of SAM were proportional to the activity of MTHFR; in diabetics, it was not. Consistent with previous observations, progression of nephropathy was accompanied by increased concentrations of tHcy. Increased erythrocyte concentrations of SAH, decreased erythrocyte concentrations of SAM, SAM/SAH ratios, and lymphocyte MTHFR activity also accompanied disease progression. The blood concentrations of SAH paralleled those of tHcy, while the concentrations of SAM showed a bimodal relationship with those of tHcy. These results provide further evidence that alterations in the blood concentrations of SAM and related compounds are abnormal in patients with diabetes, particularly in those with nephropathy. The deficiency of SAM may lead to methyl deficiencies, which may contribute to the high morbidity and mortality in patients with diabetic nephropathy. We have also demonstrated a decrease in lymphocyte MTHFR activity in patients with advanced nephropathy, suggesting that hyperhomocysteinemia in these patients may be due to a generalized metabolic abnormality. Further studies are needed to determine the pathogenesis of these abnormalities and whether they are present in renal failure due to causes other than diabetes or whether they are specific to diabetic nephropathy.

Selection and implementation for coagulation instruments/reagents in a multiple hospital/clinic network.

Selection, standardization, and implementation of instrumentation and reagents throughout a health care facility network can often be a difficult process. However, in today's ever-changing health care setting, it is often mandated. The Veteran's Integrated Systems Network 16 (VISN 16) was faced with such a task early in 1999, with the targeted area being its coagulation laboratories. The plan outlined in this paper was drafted to help facilitate the selection, standardization and implementation of coagulation systems for 17 health care facilities that make up the VISN 16 network. The VISN, encompassing 170,000 square miles, has 10 tertiary care hospitals, six of which have close relationships with affiliate universities. There are 299,733 patients enrolled in this health delivery system. The facilities range from large institutions performing both tertiary and outpatient care to small outpatient clinics. Because of the plan's detailed, comprehensive content, which included analyses of a large number of performance parameters as well as cost-efficiency, the selection process was carried out using a checklist that could be helpful to other organizations selecting equipment and reagents for coagulation studies. An implementation process was devised, resulting in coagulation standardization across the Integrated Health Network.

Hyperhomocysteinemia in type 2 diabetes mellitus: cardiovascular risk factors and effect of treatment with folic acid and pyridoxine.

OBJECTIVE: To determine whether hyperhomocysteinemia (HH) exacerbates other cardiovascular risk factors and markers of coagulation and hemostasis in patients with type 2 diabetes mellitus (DM) and whether treatment of HH with vitamins will alter these risk factors. METHODS: We measured several cardiovascular risk factors and markers of coagulation and hemostasis in patients with type 2 DM with and without HH. We also treated patients with type 2 DM and coexistent HH with high doses of folic acid and pyridoxine to determine whether this treatment would lower plasma total homocysteine concentrations as well as correct other associated cardiovascular risk factors in this population. RESULTS: Plasma levels of plasminogen activator inhibitor type 1 and fibrinogen were significantly higher in all patients with DM in comparison with control subjects (P<0.01), whether they had HH or not. No significant difference was noted between the two groups of patients with DM. The presence of hypertension and microalbuminuria did not lead to a higher plasma total homocysteine. After treatment with folic acid, 15 mg daily, and pyridoxine, 600 mg daily, fasting (basal) plasma total homocysteine declined significantly in patients with DM from 12.3 +/- 2.9 micromol/L to 9.1 +/- 1.1 micromol/L (P<0.01). The peak post-methionine load plasma total homocysteine in the patients with DM decreased from 39.9 +/- 11.4 micromol/L to 30.4 +/- 6.5 micromol/L (P<0.05). Neither fasting nor peak plasma total homocysteine changed in normal subjects. None of the cardiovascular risk factors measured changed significantly with the vitamin treatment. CONCLUSION: The coexistence of type 2 DM and HH does not lead to an exacerbation of abnormalities in the measured variables of coagulation and hemostasis. Treatment with high doses of folic acid and pyridoxine lowers the plasma total homocysteine significantly but does not improve any of the associated cardiovascular risk factors that we measured. Long-term clinical trials should be conducted to determine whether high-dose vitamin treatment will diminish the increased morbidity and mortality associated with cardiovascular disease in patients with type 2 DM.

Is testing for inherited coagulation inhibitor deficiencies in young stroke patients worthwhile?

OBJECTIVE: To test the hypothesis that in patients under age 50, with a first, arterial, ischemic cerebral infarct, whose family history and medical history do not suggest an inherited coagulation inhibitor deficiency, the yield of a laboratory search for these disorders will be low. MATERIALS AND METHODS: In 55 such patients under age 50, we systematically searched for deficiencies of protein C, protein S, and antithrombin III. RESULTS: No abnormalities of protein C or antithrombin III were found. One patient had a deficiency of protein S, which was most likely acquired rather than inherited. CONCLUSIONS: In patients who lack clinical features of a prothrombotic state, the yield of testing for protein C, S and AT III deficiency is likely to be low.

Molecular cloning and sequence analysis of the 5'-flanking region of the Sprague-Dawley rat thrombomodulin gene.

The 5'-flanking region of the rat thrombomodulin gene was cloned by polymerase chain reaction (PCR) amplification of adaptor-ligated rat genomic DNA fragment libraries, using primers derived from the coding sequences of the thrombomodulin cDNA and adaptor primers. By sequence analysis putative regulatory elements in the promoter domain were shown to include a TATA box and several conserved binding sites for stimulatory protein 1 (SP1) and activator protein 2 (AP2). The transcription factor activator protein 1 (AP1) binding site located in the 5'-flanking region may serve as a negative gene regulatory site for tumor necrosis factor-alpha (TNF-alpha). A potential retinoic acid response element (RARE) and a possible cAMP response element are located in the putative promoter region, suggesting a role for retinoic acid and cAMP in the induction of thrombomodulin gene expression. The rat thrombomodulin gene promoter sequence shows 89% homology to that of mouse and 77% homology to that of human.

Circulating thrombomodulin during radiation therapy of lung cancer.

The endothelial cell glycoprotein, thrombomodulin (TM), is an important physiological anticoagulant. TM is downregulated and released from the cell membrane into the circulation by ionizing radiation and during inflammation. The present study measured plasma TM in 17 patients before, during, and after radiation therapy of lung cancer: nine patients developed radiation pneumonitis, whereas eight matched patients did not. Plasma TM did not change significantly in patients who developed radiation pneumonitis. In contrast, patients who did not develop pneumonitis exhibited a moderate, but statistically significant, decrease in plasma TM antigen during the initial 1-2 weeks, with complete normalization towards the end of treatment. Our study suggests that decreased release of TM during the early phase of radiation therapy may be associated with reduced pulmonary toxicity. The use of plasma TM as a marker of pulmonary toxicity needs further study.

Hyperhomocysteinemia and thrombosis.

Homocysteine has been identified as an independent risk factor for atherosclerotic and thrombotic disease. Both arterial (cerebrovascular, carotid, coronary, and peripheral arterial) and veno-occlusive disease, jointly termed vascular occlusive disease (VOD) in this review, have been associated with hyperhomocysteinemia. In cases of homocystinuria, plasma homocysteine levels are markedly elevated. In this setting, the association between homocysteine and VOD seems clear. However, in cases of mild to moderate homocysteinemia, controversy remains regarding the association between homocysteine and VOD. In part this controversy occurs because VOD has multiple etiologies. Similarly, homocysteine levels are affected by several factors including vitamin status, age and gender, and genotype of the patient. The multiple etiologies of both VOD and hyperhomocysteinemia make controlled studies assessing their interrelationship difficult to perform. This review will attempt to present studies that either support or rebut homocysteine as an independent risk factor for vascular occlusive disease and will show that the study of homocysteine and thrombosis remains an active area of research.

An automated method for the analysis of T-cell receptor repertoires. Rapid RT-PCR fragment length analysis of the T-cell receptor beta chain complementarity-determining region 3.

The examination of T-cell receptor (TCR) repertoires has an important role in the study of lymphoproliferative disorders and autoimmune diseases. Analysis of the complementarity-determining region 3 (CDR3) of the TCR beta chain is used to assess the clonality of T-cell populations. We developed a rapid fluorescence-based method for CDR3 length analysis of expressed TCR gene families. TCR beta chain complementary DNA is amplified by a nested polymerase chain reaction with V beta family-specific oligonucleotide primers and a fluorochrome-labeled C beta primer. The polymerase chain reaction products were analyzed on a compact automated DNA sequencing system (OpenGene system, Visible Genetics, Toronto, Ontario). To demonstrate the usefulness of our technique, we examined the CDR3 length distribution of peripheral blood T cells from a healthy subject, intestinal T cells from a patient with ulcerative colitis, and the T-cell leukemia cell line Jurkat. The analysis revealed polyclonal, oligoclonal, and monoclonal CDR3 distributions, respectively, for the 3 T-cell populations. Our new method shows virtually identical CDR3 length patterns compared with the traditional radioisotope-based method. The new technique offers the convenience of rapid throughput, nonradioactive labeling, and quality data analysis.

cDNA cloning and sequencing, gene expression, and immunolocalization of thrombomodulin in the Sprague-Dawley rat.

Thrombomodulin (TM), in addition to its significance in the protein C anticoagulant pathway and cardiovascular diseases, has recently been shown to play important roles in normal embryonic development, several inflammatory conditions, as well as in tumor biology and in the pathogenesis of chronic radiation toxicity. We cloned and sequenced the cDNA encoding the complete TM protein from the Sprague-Dawley rat. The cDNA sequence consisted of a 78-bp 5' non-coding region and a 1731-bp open reading frame encoding 577 amino acids. Comparison of the deduced amino acid sequences showed Sprague-Dawley rat TM to be 87% homologous with mouse and 70.3% with human TM. In addition to the previously described highly conserved region in the lectin-like domain, another region was found which possessed significant homology among the species and may be involved in regulating cell surface expression of TM. Primers and fluorogenic probe for 5' exonuclease-based real time RT-PCR detection (TaqMan PCR) were constructed based on the cDNA sequence information and used to determine steady-state TM mRNA levels in lung, intestine, kidney, brain, and liver. The highest TM mRNA levels were found in lung and the lowest in liver. Immunohistochemistry confirmed that TM was mainly localized on the endothelium of blood vessels and lymphatics. The alveolar capillaries of lung showed the strongest immunoreactivity, whereas the endothelium of hepatic sinusoids and cerebral cortex were virtually negative.

Plasma homocysteine concentrations in type II diabetic patients in India: relationship to body weight.

Hyperhomocysteinemia has been established as a risk factor for cardiovascular disease and occurs with a high prevalence in patients with type II diabetes and microvascular disease. In order to determine whether plasma homocysteine concentrations vary with body-mass index in patients with type II diabetes, we measured plasma homocysteine in lean, normal weight, and overweight subjects living in India. Plasma homocysteine concentrations were significantly lower in the lean persons with diabetes when compared to those who were obese and compared to control subjects (p < 0.02). We conclude that plasma homocysteine concentrations are lower in lean persons with type II diabetes and that this efficiency in homocysteine metabolism may contribute towards protection from cardiovascular disease in this population.

Hyperhomocysteinemia increases intimal hyperplasia in a rat carotid endarterectomy model.

PURPOSE: This preliminary study investigated the ability to elevate the serum homocysteine (H[e]) levels and investigated the increases in postoperative neointimal hyperplasia (IH) in an environment with hyperhomocysteinemia and the resultant restenosis in a rat carotid endarterectomy (CEA) model. METHOD: The 9 rats for the control group were fed rat chow, and the 8 rats for the H(e) group were fed H(e)-supplemented rat chow for 2 weeks before and after CEA. The animals underwent anesthesia, and a left common CEA was performed. After 14 days, the serum H(e) levels were measured and the left carotid artery was harvested and elastin stained. Morphometric measurements were used to calculate the area of stenosis of the lumen. The mean and the standard deviation of the mean were determined. The 2 groups were compared with the Mann-Whitney test and a linear regression model. Three additional rats per group were studied, with carotid artery sectioning with double immunohistochemical staining for 5-bromodeoxyuridine (BrdU) and alpha-smooth muscle (alpha-SM) actin. RESULTS: The serum H(e) level in the H(e) group was 36.32 micromol/L +/- 15.28, and in the control group the level was 5.53 micromol/L +/- 2.06 (P =.0007). IH presented as percent lumen stenosis was 21.89% +/- 4.82% in the H(e) group and 4.82% +/- 1.64% in the control group (P =.0007). The linear regression model of the serum H(e) levels and the percent stenosis showed a linear relationship (r2 =.72). The alpha-SM actin staining revealed that nearly all of the cells in the IH area were of smooth muscle or myofibroblast origin and that 10.1% +/- 2.6% of the cells were stained for BrdU in the control group versus 23% +/- 7.1% in the H(e) group. Also, 9.3% +/- 2.6% of the cells in the IH area were stained for BrdU and for alpha-SM actin versus 19.1% +/- 5. 6% stained for both BrdU and alpha-SM actin in the H(e) group. CONCLUSION: This is the first study to examine IH after CEA and hyperhomocysteinemia in rats. The study shows that the elevation of serum H(e) levels can be obtained by feeding rats modified diets with added H(e). The consistent elevation of serum H(e) levels was associated with more than 4 times the amount of IH after a CEA in a rat model.