Neovascularization of the amniotic membrane as a biological immune barrier.

The clinical application of islet transplantation is limited due to the limited source and the morbidity of systemic immunosuppression to prevent rejection. The two problems can be solved by using encapsulated islets. We have used amniotic membranes as biocompatible natural immune barriers. The objective of this study was to assess the revascularization of the membrane, which is necessary to ensure islet viability when the membrane is used for islet encapsulation. The amniotic membranes, obtained from full-term pregnant female dogs, were molded to form macrocapsules, which were implanted in the peritoneal cavity. The capsules were removed after 3, 10, 15, and 30 days and examined histopathologically using hematoxylin and eosin and by immunohistochemistry for neovascularization using factor VIII to detect angiogenesis. Upon histopathological examination, all specimens showed localized, moderate inflammation and congested blood vessels with no thrombosis or rejection. There was a mild degree of fibroblast proliferation starting from day 10 to day 30. Immunohistochemical staining revealed that the number of blood vessels was 7, 11, 13, 10 per high-power microscopic field on days 3, 10, 15, and 30, respectively. We concluded from this study that implanted amniotic sac capsules were vascularized within the omental tissue from day 10 on with significant blood vessel formation starting on day 3 by immunohistochemical study.

Myocardial protection with monophosphoryl lipid-A against aortic cross clamping-induced global stunning.

BACKGROUND: Monophosphoryl lipid-A (MLA) has a late window (24 hours) of cardioprotection against acute myocardial infarction. It is not known whether MLA, administered, 24 hours before surgery, attenuates intraoperative ventricular dysfunction "stunning" associated with aortic cross-clamping and reperfusion during elective cardiac surgery. We determined the dose-response relationship between MLA and ventricular function in a canine model of global myocardial stunning in the absence of necrosis. The role of expression of inducible heat shock protein 70 (HSP 70i) was also investigated. METHODS: Mongrel dogs (n = 32) were intravenously injected with either a vehicle solution or 3, 5, 10, 35 ug/kg MLA. Twenty four hours later, dogs were anesthetized and instrumented, in situ, to monitor the left ventricular performance (the slope of regression between stroke-work and end diastolic length). Tissue samples were obtained to determine HSP70i using immunoblot analysis. After a period of equilibration on cardiopulmonary bypass, the aortic cross-clamp was applied at normothermia for 30 minutes followed by 60 minutes of reperfusion. ATP and catabolites were determined in transmural myocardial biopsies. Triphenyl-tetrazolium chloride (TTC) staining was used to determine myocardial necrosis. RESULTS: MLA treatment did not alter myocardial contractility or ATP metabolism. Global ischemia resulted in about 50% depletion of ATP and remained depressed during reperfusion in all groups. MLA-treated hearts had improved functional recovery in a dose dependent-manner. Significant recovery was observed at the highest dose (35 ug/kg) compared to the control group. Immunoblot analysis demonstrated significant increase in HSP 70i in the MLA-treated hearts. CONCLUSIONS: MLA exhibits a delayed (24 hours) window of protection against myocardial stunning associated with aortic cross-clamping. HSP70i expression may play a role in MLA-mediated cardioprotection.

Hyperdynamic circulation of arteriovenous fistula preconditions the heart and limits infarct size.

BACKGROUND: Chronic arteriovenous fistulae (AVF) create sustained hyperdynamic circulation. It is not known whether hyperdynamic circulation alters myocardial sensitivity to ischemia and reperfusion injury. We tested the hypothesis that AVF activate molecular responses that increase tolerance to infarction in dogs. METHODS: Twelve dogs were divided into two groups: 1) AVF group, where an AVF in the femoral region was done; and 2) sham-operated group (each n = 6). After 8 weeks, left ventricular performance was determined from stroke work/end-diastolic length relationship. Myocardial biopsy was obtained to determine heat-shock protein 70 and adenosine triphosphate (ATP) pool. Left anterior descending coronary artery was occluded for 90 minutes at 37 degrees C, followed by 4 hours of reperfusion. Coronary blood flow was determined using different colored microspheres. RESULTS: The fistula group showed improvement of left ventricular performance (p = 0.03). The infarct size was significantly lower in the fistula group; it was 9.2+/-2.0% in the fistula group versus 28.4+/-5.2% in the sham group (p < 0.05). ATP depletion during ischemia was less in the fistula group (p = 0.02). Regional myocardial blood flow was significantly higher in the fistula group (p = 0.03). CONCLUSIONS: Peripheral AVF improve the left ventricular performance, and decrease infarct size and ATP depletion. This protective effect is caused by the development of collaterals in the coronary circulation without expression of heat-shock protein 70.

Redox regulation of signal transduction in vascular smooth muscle cells: thiol oxidizing agents induced phospholipase D.

Decrease in intracellular thiols leads to oxidative stress and thus may cause alterations in the activity of redox-sensitive enzymes required for signal transduction. Here, we report that, N-ethylmaleimide and phenylarsine oxide, which are known to oxidize free thiols as well as protein thiols, induced phosphatidyl ethanol generation in the micromolar range suggesting activation of phospholipase D in vascular smooth muscle cells. These agents also induced significant phosphatidic acid and diacylglycerol generation without causing protein kinase C activation. Phenylarsine oxide and N-ethyl maleimide induced phospholipase D activation is protein kinase C independent as it was not inhibited by compound-3 and bisindolylmaleimide, potent protein kinase C inhibitors. Tyrosine kinase inhibitor herbimycin A by itself activated PLD, but inhibited the phospholipase D activation by phenylarsine oxide and N-ethylmaleimide. These results suggest that oxidation of the cellular thiols activates phospholipase D independent of protein kinase C.

Redox regulation of signal transduction in smooth muscle cells: distinct effects of PKC-down regulation and PKC inhibitors on oxidant induced MAP kinase.

Reactive oxygen species function as signaling molecules, and are known to be generated under both normal and pathological conditions. Using vascular smooth muscle cells, we have demonstrated an increase in mitogen activated protein kinase activity in response to oxidants. Mitogen activated protein kinase activity increased linearly with time in cells treated with pervanadate. Hydrogen peroxide also caused rapid induction of mitogen activated protein kinase. Protein kinase C down regulation partially decreased induction of mitogen activated protein kinase activity by oxidants, and the Ca2+ ionophore, ionomycin. Protein kinase C inhibitors, compound-3 and bisindolylmaleimide did not inhibit oxidant induced mitogen activated protein kinase activity, where as calphostin C activated it. The tyrosine kinase inhibitors genistein, herbimycin A and tyrphostin caused 50% inhibition of oxidant induced mitogen activated protein kinase activation. These results suggest that oxidant-induced mitogen activated protein kinase is protein kinase C independent.

Diabetes mellitus and cardiac function.

Cardiovascular complications are the most common causes of morbidity and mortality in diabetic patients. Coronary atherosclerosis is enhanced in diabetics, whereas myocardial infarction represents 20% of deaths of diabetic subjects. Furthermore, re-infarction and heart failure are more common in the diabetics. Diabetic cardiomyopathy is characterized by an early diastolic dysfunction and a later systolic one, with intracellular retention of calcium and sodium and loss of potassium. In addition, diabetes mellitus accelerates the development of left ventricular hypertrophy in hypertensive patients and increases cardiovascular mortality and morbidity. Treating the cardiovascular problems in diabetics must be undertaken with caution. Special consideration must be given with respect to the ionic and metabolic changes associated with diabetes. For example, although ACE inhibitors and calcium channel blockers are suitable agents, potassium channel openers cause myocardial preconditioning and decrease the infarct size in animal models, but they inhibit the insulin release after glucose administration in healthy subjects. Furthermore, potassium channel blockers abolish myocardial preconditioning and increase infarct size in animal models, but they protect the heart from the fatal arrhythmias induced by ischemia and reperfusion which may be important in diabetes. For example, diabetic peripheral neuropathy usually presents with silent ischemia and infarction. Mechanistically, parasympathetic cardiac nerve dysfunction, expressed as increased resting heart rate and decreased respiratory variation in heart rate, is more frequent than the sympathetic cardiac nerve dysfunction expressed as a decrease in the heart rate rise during standing.

Correlation between the level of serum growth hormone; somatomedin-C; anthropometric measurements and the liver functions in chronic liver diseases.

Twenty patients with bilharzial periportal fibrosis (group I); 20 patients with post hepatitic cirrhosis (group II) and 20 normal controls were chosen to study the correlation between the serum level of human growth hormone (GH) and Somatomedin-C (Sm-C); and the anthropometric measurements; liver function tests and various haematological parameters. Group I showed no statistically significant difference in the mean values of serum GH before and after insulin induced hypoglycemia. The basal serum level of Sm-C showed a statistically significant decrease and highly significant decrease after insulin test. A statistically significant positive correlation was evident between the individual values of serum level of GH and Sm-C and the anthropometric measurements, haematological values and liver function tests (r = 0.41). The mean serum level of GH in group 2, showed statistically significant increase before and after insulin test. The mean serum level of Sm-C showed a statistically significant decrease before and after insulin test. A statistically significant negative correlation was found between the individual values of the serum level of GH and Sm-C and the anthropometric measurements, haematological values and liver function tests (r = 0.16).